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WO1996018733A2 - Ribozyme-mediated inactivation of leukemia-associated rna - Google Patents

Ribozyme-mediated inactivation of leukemia-associated rna Download PDF

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WO1996018733A2
WO1996018733A2 PCT/US1995/016451 US9516451W WO9618733A2 WO 1996018733 A2 WO1996018733 A2 WO 1996018733A2 US 9516451 W US9516451 W US 9516451W WO 9618733 A2 WO9618733 A2 WO 9618733A2
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rna
apl
group
rarα
ribozyme
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PCT/US1995/016451
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WO1996018733A3 (en
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Umberto Pace
Shaji T. George
Allan R. Goldberg
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Innovir Laboratories, Inc.
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Priority to AU49619/96A priority Critical patent/AU4961996A/en
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Publication of WO1996018733A3 publication Critical patent/WO1996018733A3/en

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Definitions

  • This application is directed to methods and ribozyme and antisense oligonucleotides compositions designed to inactivate RNA molecules associated with malignancies arising from chromosomal translocations, especially specific leukemias, such as Acute Promyelocytic Leukemia (APL) .
  • APL Acute Promyelocytic Leukemia
  • Acute Promyelocytic Leukemia About 10% of acute myeloblastic leukemias
  • a in adults is acute promyelocytic leukemia (APL, French American British Classification (FAB) M3), see arrell, R.P., et al . , New England J. Med. , 329, 177-189 (1993) for reviews) .
  • the disease typically presents with a bleeding diathesis which is often exacerbated by chemotherapy, leading to a high rate of early mortality, primarily from intracranial hemorrhage.
  • the bleeding diathesis is due to the presence of malignant promyelocytes which release procoagulant substances. These, in turn, activate the coagulation cascade, depleting fibrinogen, clotting factors and platelets.
  • APL is consistently associated with a non- random chromosomal abnormality, characterized by a balanced and reciprocal translocation between the long arms of chromosomes 15 and 17 (t(15;17)) , found in over 90% of patient-derived APL cells (Kakizuka, A., et al .
  • the fusion product, PML-RAR ⁇ displays altered transactivating properties compared with wildtype RAR ⁇ gene product, which acts as a transcription enhancer in response to retinoic acid (RA) (Kakizuka, A., et al., Cell , 66, 663-674, (1991) ; de The, H., et al . , Cell , 66 , 675- 684 (1991); Pandolfi, P.P., et al . , Oncogene, 6, 1285-1292 (1991)) . It has been shown that ATRA induces maturation of the leukemia cells both in vivo (Warrell, R.P., et al . , New England J. Med .
  • RA retinoic acid
  • PML-RAR ⁇ functions as a dominant negative mutation, its product blocking myeloid differentiation.
  • Evidence for the involvement of the PML-RAR ⁇ protein in the pathogenesis of APL is provided by its expression in U937 cells, which results in a block in differentiation, increased sensitivity to RA, and increased cell survival in the presence of limiting serum in the culture media (Grignani, F., et al . , Cell , 74, 423-431, (1993)) .
  • the bcr2 region spans a region encompassing a small portion of intron 4, exon 5, intron 5 and exon 6 of PML. Translocations involving this breakpoint are essentially different from one another and many of them occur inside PML exons, causing a large variation in the fusion sequences and, occasionally, generating aberrant reading frames, which code for aberrant and truncated proteins.
  • the J cr3 region is located in intron 3 of PML and invariably results in a mRNA in which exon 3 of PML and exon 3 of RAR ⁇ are spliced together. The sequence in the fusion junction is identical in all the J cr3 cases.
  • bcrl and Jbcr3-type junctions account for at least 80 percent of the tested APL cases (Pandolfi, P.P., et al . , EMBO J. , 11, 1397-1407 (1992)) , with one study finding Jbcrl-type junctions at twice the rate of J cr3-type ones (Miller, W.H., Jr., et al . , Proc . Na tl . Acad. Sci USA, 89, 2694-2698 (1992)) .
  • Other Translocational Cancers Many other cancers have been reported in the literature as arising due to, or associated with, chromosomal translocations.
  • Examples include RBTN2 and t[ll; 14] [pl3 ; qll] in T cell acute leukemia and erythropoiesis, translin in lymphoid neoplasms, T[5;14] [q34;qll] in acute lymphoblastic leukemia, T14;18 chromosomal translocations in follicular lymphoma, Non-Hodgkin' s lymphoma, Hodgkin's disease; T18 translocations in human synovial sarcomas; Burkitt's lymphoma; t[ll; 22] [q24 ; ql2] translocation in Ewing sarcoma; t[3p; 6p] and t [12q; 17p] translocations in human small cell lung carcinomas; and t[15; 19] translocation in diseminated mediastinal carcinoma.
  • the transcription product of the fusion or the fusion itself represent targets for therapy, if a
  • an object of the present invention to provide molecules and methods for treating patients or cells derived from patients having cancerous cells arising from or characterized by chromosomal translocations. It is a further object of the present invention to provide molecules and methods for treating patients or cells derived from leukemia patients that inactivate specific cancer-associated RNA, including APL-associated RNA produced in the affected blood cells of leukemia patients.
  • RNA molecules such as ribozymes and external guide sequence (EGS) molecules for RNAse P, are engineered to promote efficient and specific ribozyme cleavage of mRNA associated with various types of cancers, especially leukemia-associated mRNA, including mRNA associated with APL.
  • Antisense molecules are also designed which are directed against specific cancer-associated mRNA to promote inhibition of its expression.
  • Engineered RNA molecules are designed and synthesized which contain specific nucleotide guide sequences which enable a ribozyme or external guide sequence for RNAse P to preferentially bind to and promote ribozyme-mediated cleavage of a specific cancer- associated RNA, or to block transcription.
  • RNAse P examples demonstrate that ribozymes and EGS molecules for RNAse P have been constructed that bind to and promote ribozyme cleavage of leukemia- associated RNA in cells. Methods for the determination of the activity of a ribozyme or an EGS for the purpose of construct-screening, as well as methods for using and producing such RNA molecules, are also disclosed.
  • Figure 1 is a schematic of the proposed intra- and interstrand complementary base binding structures formed by ribozyme IHRZ1.18 (SEQ ID NO. 5) with (a) a PML-RAR ⁇ substrate RNA molecule (nucleotide (nt) 1721 to 1754 of SEQ ID NO. 3) and (b) with a RAR ⁇ substrate RNA molecule (corresponding to nt 142 to 175 of SEQ ID No. 4) .
  • the substrate RNA nucleotide sequence is shown in boldface characters.
  • An asterisk indicates the location of the nucleotide which corresponds to the site in RAR ⁇ mRNA which becomes fused to PML mRNA nucleotides by transcription of PML-RAR ⁇ gene fusions .
  • the cleavage sites are indicated by arrows.
  • Figure 2 is a schematic of the proposed intra- and interstrand complementary base binding structures formed by ribozyme IHRZ1.3 (SEQ ID NO. 6) with (a) a PML-RAR ⁇ and (b) a RAR ⁇ substrate RNA molecules.
  • the substrate RNA nucleotide sequence is shown in boldface characters.
  • An asterisk indicates the location of the nucleotide which corresponds to the site in RAR ⁇ mRNA which becomes fused to PML mRNA nucleotides by transcription of PML-RAR ⁇ gene fusions.
  • the cleavage sites are indicated by arrows.
  • Figure 3 is a schematic of the proposed intra- and interstrand complementary base binding structures formed by ribozyme IHRZ1.30 (SEQ ID NO. 7) with (a) a PML-RAR ⁇ substrate molecule (nt 1701 to 1754 of SEQ ID NO. 3) and (b) a RAR ⁇ substrate RNA (nt 136 to 175 of SEQ ID NO. 4) molecule.
  • Figure 4 is a graph of percent substrate RNA molecules not cleaved as a function of ribozyme concentration ( ⁇ m) as determined by in vi tro cleavage assay of PML-RAR ⁇ (closed circles) and RAR ⁇ (open circles) mRNA substrate molecules by IHRZ1.18. Each point is an average of three experiments.
  • Figure 5 is a graph of percent substrate RNA molecule not cleaved as a function of ribozyme concentration ( ⁇ m) as determined by in vi tro cleavage assay of PML-RAR ⁇ (closed circles) and RAR ⁇ (open circles) mRNA substrate molecules by IHRZl.3. Each point is an average of three experiments.
  • Figure 6 is a graph of percent substrate RNA molecule not cleaved as a function of ribozyme concentration ( ⁇ m) as determined by in vi tro cleavage assay of PML-RAR ⁇ (closed circles) and RAR ⁇ (open circles) mRNA substrate molecules by IHRZl.30. Each point is an average of three experiments.
  • Figure 7 is a schematic of a nuclease- resistant ribozyme designed to have all non-core nucleotides replaced with 2' 0-methyl ribonucleotides or phosphorothioate deoxyribonucleotides. Unmodified core sequence nucleotides shown in italics.
  • Figure 8 is a diagram of a ribozyme expression vector.
  • Figure 9 is the structure and sequence of anti-APL hammerhead ribozyme constructs (SEQ ID NO. 12) targeted to APL transcripts (nt. 1-41 of SEQ ID NO. 13) .
  • the underlined nucleotides are found in the .1 series. Changes in the 5.n series
  • active control are indicated: changed to C in 5.0 and 5.1, deleted in 5.0 and 5.1.
  • Figure 10 is the structure and sequence of anti-APL hammerhead ribozyme constructs (SEQ ID NO. 14) targeted to APL transcripts (nt. 3-50 of SEQ ID NO. 13) .
  • the underlined nucleotides are found in the .1 series. Changes in the 6.n series (inactive control) are indicated: changed to C in 6.0 and 6.1, deleted in 6.0 and 6.1.
  • Figure 11 is a graph of the MTT assay for inhibition of cell growth, plotting optical density (i.e., cell number) over time (days) for cells exposed to APL 2.0, 65 ⁇ g/ml of hygromycin (dark squares) ; APL 2.0 500 ⁇ g/ml of hygromycin (open squares) ; APL 2.1 65 ⁇ g/ml of hygromycin (dark diamonds) ; APL 2.1 500 ⁇ g/ml of hygromycin (open diamonds) ; APL 5 65 ⁇ g/ml of hygromycin (dark triangles) ; APL 5 500 ⁇ g/ml of hygromycin (open triangles) .
  • optical density i.e., cell number
  • Figure 12 is a graph of the MTT assay for inhibition of cell growth, plotting optical density (i.e., cell number) over time (days) for cells exposed to APL 2.1 in combination with various concentrations of hygromycin: 65 ⁇ g/ml of hygromycin (dark squares) ; 130 ⁇ g/ml of hygromycin (open squares) ; 195 ⁇ g/ml of hygromycin (dark diamonds) ; 260 ⁇ g/ml of hygromycin (open diamonds) ; 325 ⁇ g/ml of hygromycin (dark triangles) ; 490 ⁇ g/ml of hygromycin (open triangles) .
  • Figures 13a, 13b, 13c, and 13d are the structures and sequences of external guide sequences targeted to the fusion junction of PML RAR.
  • Figure 13a is APL EGS A20 (APL RNA is nt. 7- 24 of SEQ ID NO. 13; EGS RNA is SEQ ID NO. 15) ;
  • Figure 13b is the inactive control A20D (SEQ ID NO. 15 minus nt 22 and 23);
  • Figure 13c is the APL EGS
  • APL RNA is nt. 6-22 of SEQ ID NO. 13; EGS RNA is SEQ ID NO. 16) ; Figure 13d is the inactive control 1017 (SEQ ID NO. 15 minus nt 14, 17, 18, 29) .
  • Figures 14a and 14b are graphs of the MTT assay for inhibition of cell growth, plotting optical density (i.e., number of cells) over time (days) , for APL target EGS A20 ( Figure 14a) and inactive control EGS ( Figure 14b) at concentrations of 10 ⁇ M (dark square) , 9 ⁇ M (open square) , 8 ⁇ M (dark diamond) , 7 ⁇ M (open diamond) , 6 ⁇ M (dark triangle) , 5 ⁇ M (open triangle) , 4 ⁇ M (dark circle) , 3 ⁇ M (open circle) , 2 ⁇ M (X) , and 1 ⁇ M (*) .
  • Figures 15a and 15b are graphs of the MTT assay for inhibition of cell growth, plotting optical density (i.e., number of cells) over time (days) , for APL target EGS 1009 ( Figure 15a) and inactive control EGS ( Figure 15b) at concentrations of 10 ⁇ M (dark square) , 9 ⁇ M (open square) , 8 ⁇ M (dark diamond) , 7 ⁇ M (open diamond) , 6 ⁇ M (dark triangle) , 5 ⁇ M (open triangle) , 4 ⁇ M (dark circle) , 3 ⁇ M (open circle) , 2 ⁇ M (X) , and 1 ⁇ M (*) •
  • RNA molecules suitable for use in the treatment of cancers associated with chromosomal translocations have been designed.
  • the RNA molecules are ribozymes or external guide sequences specifically binding to and cleaving RNA in specific leukemias, such as those of APL. I. Determination of Specific Cancer-Associated RNA Sequence.
  • ribozymes, EGSs, or antisense oligonucleotides can be designed which block transcription of the translocated RNA.
  • Oligonucleotides are designed based on the same principles for these cancers as for APL RNA.
  • APL-associated mRNA molecules encoding proteins associated with APL are formed by transcription of aberrant gene fusions characteristically found in the particular type of APL. Accordingly, the presence of specific APL- associated mRNA must first be identified.
  • nucleotide sequence data of that mRNA must be obtained so that ribozymes or EGS molecules can be engineered to bind to one or more nucleotide sequences uniquely characteristic of the particular APL-associated mRNA.
  • the leukemic promyelocytes of a patient with APL contain RNA transcripts of the specific PML-RAR ⁇ chromosomal translocation characteristic of that patient's type of APL. Accordingly, the presence of cancer-associated mRNA must be identified in a patient, and its sequence determined in order to design and use the ribozymes, EGS molecules and antisense oligonucleotides described herein to preferentially cleave, and thereby inactivate, the specific cancer-associated mRNA.
  • Diagnosis of APL can be made using histopathological data, cytogenetic data and polymerase chain reaction (PCR) analysis.
  • the PCR data should yield the kind of breakpoint present at the PML-RAR ⁇ junction.
  • More than 80% of the APL cases have either a bcr-1 or a Jbcr-3-type junction. In these cases, there is no need to design a new ribozyme or EGS, but one of the constructs described herein can be used, either presynthesized or cloned into a vector.
  • the junction is a j cr-2-type, if a ribozyme or EGS molecule is not available, one can be designed and synthesized as described below.
  • the identification of the various characteristic Jbcr sequences makes the typing of a particular APL routine using standard methods.
  • the characteristic Jbcr sequences of the various types of APL can be used as probes in standard hybridization blots to identify PML-RAR ⁇ RNA transcripts or PML-RAR ⁇ gene fusions, and the nucleotide sequence of such PML-RAR ⁇ RNA or DNA molecules can be routinely determined by standard cloning and nucleic acid sequencing methods (see, for example, Sambrook et al., In Molecular Cloning: A Laboratory Manual, second edition: Vol. 1: 7.39- 7.87 (RNA hybridization and sequence analysis) ;
  • Ribonucleic acid (RNA) molecules can serve not only as carriers of genetic information, for example, genomic retroviral RNA and messenger RNA (mRNA) molecules and as structures essential for protein synthesis, for example, transfer R ⁇ A (tR ⁇ A) and ribosomal R ⁇ A (rR ⁇ A) molecules, but also as enzymes which specifically cleave nucleic acid molecules.
  • mRNA messenger RNA
  • tR ⁇ A transfer R ⁇ A
  • rR ⁇ A ribosomal R ⁇ A
  • Such catalytic RNA molecules are called ribozymes .
  • ribozymes include certain RNA sequences known as intervening sequences or introns. These sequences are removed in order to generate the final functional RNA. It was shown that many members of two classes of introns, groups I and II, ubiquitously found in lower eukaryotes, can excise themselves without the help of protein factors. This kind of ribozyme was discovered by Thomas Cech and colleagues, who have discussed some in vitro applications (see, in PCT/US887/03161, published as WO 88/04300 16 June 1988, see also, Cech, T. , Annu. Rev. Biochem. , 59, 543-568, (1990) ) .
  • RNAse P transfer RNA
  • Bacterial RNase P includes two components, a protein (C5) and an RNA (Ml) .
  • C5 protein
  • Ml RNA
  • Altman and colleagues developed a method for converting virtually any RNA sequence into a substrate for bacterial RNase P by using an external guide sequence (EGS) , having at its 5' terminus at least seven nucleotides complementary to the nucleotides 3' to the cleavage site in the RNA to be cleaved and at its 5' terminus the nucleotides NCCA (N is any nucleotide) (Forster, A.C. and Altman, S., Science, 238, 407-409 (1990)) .
  • EGS external guide sequence
  • EGS/RNase P-directed cleavage of RNA has been developed for use in eukaryotic systems, (Yuan, Y., Hwang, E.S., and Altman, S., Proc . Natl . Acad . Sci . USA, 89, 8006-8010 (1992)) .
  • These external guide sequences have more stringent requirements, however.
  • the EGSs contain sequences which are complementary to the target RNA and which forms secondary and tertiary structure akin to portions of a tRNA molecule.
  • a eukaryotic EGS must contain at least seven nucleotides which base pair with the target sequence 3' to the intended cleavage site to form a structure like the amino acyl acceptor stem, nucleotides which base pair to form a stem and loop structure similar to the T stem and loop, followed by at least three nucleotides that base pair with the target sequence to form a structure like the dihydroxyuracil stem.
  • the EGS can be made more resistant to nuclease degradation by including chemically modified nucleotides or nucleotide linkages.
  • the external guide sequence and the RNase P catalytic RNA can be used together as separate molecules.
  • the two sequences can be combined into a single oligonucleotide molecule possessing both targeting and catalytic functions.
  • a combined oligonucleotide termed an RNase P internal guide sequence (RIGS)
  • RIGS RNase P internal guide sequence
  • VLP Viroid-Like Pathogens
  • a third class of ribozymes is derived from the so-called viroid-like pathogens (VLP) , a group of self-replicating RNAs that include some plant pathogens such as viroids, virusoids and satellites of plant viruses and the hepatitis delta virus (HDV) , a human pathogen that functions as a satellite of hepatitis B virus.
  • VLP viroid-like pathogens
  • HDV hepatitis delta virus
  • a key element of the life cycle of these pathogens is their replication strategy that involves synthesis of multimeric strands of both polarities (Branch, A.D. and Robertson, H.D., Science, 223, 450-455 (1988)) .
  • multimeric units are then cleaved into monomeric units by a self-cleavage activity, present in a specific region of the sequence.
  • sequences maintain their cleaving activity once they are separated from the bulk of the sequence and can also be engineered to cleave other sequences.
  • VLP-derived ribozymes known so far belong to the "hammerhead" subclass (Sy ons R.H., Annu. Rev. Biochem. , 61, 641-671 (1992) ; Forster, A.C, and Symons, R.H., Cell , 49, 211-220, (1987) ; Uhlenbeck, O . C . , Na ture, 328, 596-600 (1987) ; Haseloff, J., and Gerlach, W.L., Nature, 334, 585- 591 (1988)) .
  • ribozyme derived from the antigenomic strand of the satellite of the tobacco ringspot virus, belongs to the "hairpin” subclass (Symons R.H., Annu. Rev. Biochem. , 61, 641-671 (1992) ; Hampel, A., Tritz, R., Biochemistry 28, 4929-4933 (1989) ) and two, derived from the genomic and the antigenomic stands of HDV are members of a third subclass, called "axehead" (Branch, A.D. and Robertson, Proc . Natl . Acad. Sci . USA, 88, 10163- 10167 (1991) ) .
  • Antisense molecules are usually single stranded DNA or RNA molecules, or their substituted analogues, which can bind to the target RNA through Watson and Crick base pairing and prevent the translation of these RNAs (Mizuno, T. , et al., Proc. Na tl . Acad. Sci . USA, 81, (1983) ; Zamecnik, in Prospects for Antisense Nucleic Acid Therapy of Cancer and Aids, ed. , Wickstrom, Wiley-Liss, New York) ) . They are usually 15 to 30 nucleotides long and have been used widely to inhibit expression of various proteins (Zamecnick, P.C. and Stevenson, M.L. Proc . Natl . Acad . Sci . , USA, 75, 280 (1978) ;
  • DNA based antisense can also inhibit expression of proteins by presenting the DNA-RNA hybrid as a target for cleavage by the endogenous RNaseH enzyme (Giles, R.V. and Tidd, D.M., Nucleic Acid Res . , 20, 763 (1992)) , thereby destroying the target RNA.
  • the antisense molecules can be made more resistant to nucleases by introducing phosphorothioate diester linkages instead of the phosphodiester linkage (Agrawal, S., et al . , Proc . Na tl . Acad . Sci . , USA, 85, 7089, (1988) ) and duplexes of these molecules with an RNA is recognized by RNaseH.
  • ribozymes and EGS molecules can be designed and synthesized which preferentially hybridize to the characteristic cancer-associated mRNA sequence and promote ribozyme-mediated cleavage or blockage of transcription or translation of that mRNA.
  • the leukemia cells contain mRNA transcripts of the PML-RAR ⁇ gene fusion characteristic of that particular type of APL.
  • the basic strategy for designing ribozymes or EGS molecules efffective against a particular type of APL is to engineer into ribozymes, EGS molecules, or antisense oligonucleotides, specific nucleotide guide sequences complementary to both sides of the particular PML-RAR ⁇ gene fusion.
  • Such engineered nucleotide guide sequences enable the ribozymes and EGS molecules to preferentially bind specific cancer-associated mRNA molecules and promote the subsequent ribozyme cleavage of, or block transcription of, the mRNA molecules.
  • an engineered ribozyme or EGS to promote ribozymal activity is readily determined using an in vi tro assay for a ribozyme's activity against a specific cancer-associated mRNA sequence, as described in more detail below.
  • the assay permits one to compare the efficiency of ribozymal cleavage against a particular PML-RAR ⁇ mRNA sequence, characteristic of that type of APL, with an unaltered wild-type RAR ⁇ mRNA sequence found in normal cells.
  • RAR ⁇ retinoic acid receptor ⁇
  • plasmids encoding portions of the PML-RAR ⁇ gene and the RAR ⁇ gene, were synthesized. These plasmids allow the synthesis of shortened versions of APL mRNA molecules in vi tro, facilitating the testing and screening process.
  • NB4 cells have been derived from APL patients (Lanotte, et al . , Blood, 77:1080 (1991)) . These cells possess a t(15;17) translocation ⁇ bcrl - type) , and they respond to treatment with ATRA, just as observed in cells of APL patients.
  • ribozymes and EGS molecules can be synthesized by transcribing DNA templates, for example, with T7
  • RNA polymerase (Milligan, et al . , Nucl Acids Res . ,
  • An in vi tro cleavage assay which measures the percentage of substrate RNA remaining after incubation with various amounts of an engineered ribozyme or EGS, in the presence of a non-limiting amount of RNAse P, is used as an indicator of the potential anti-leukemic activity of the ribozyme or the EGS/RNase P complex. Ribozymes or EGS/RNase P that exhibit the highest in vi tro activity are selected for further testing. The percentage of RNA remaining can be plotted as a function of the ribozyme (or EGS) concentration. The catalytic efficiency of a ribozyme can be expressed as k ⁇ /K,.
  • ribozyme or EGS constructs are those which bind to and promote the preferential ribozyme cleavage of the cancer-associated substrate mRNA. Preferred constructs can be selected using the ribozyme cleavage assay, as shown by Example 1, and determining which constructs are the most efficient at specifically cleaving the cancer-associated substrate RNA sequence as determined by the value of , as described above.
  • a more preferred ribozyme construct can be selected as the ribozyme which has the highest value of the ratio of the efficiency of ribozyme-mediated cleavage of the cancer-associated mRNA sequence and the efficiency of ribozyme cleavage of the related wild-type mRNA sequence found in normal cells, that is, (cancer-associated RNA) -. (wild-type RNA) .
  • the more preferred ribozyme construct is one having the highest ratio of k ⁇ /i. (PML-RAR ⁇ R ⁇ A) :k cat /K,. (wildtype RAR ⁇ R ⁇ A) (see Example 3, below) .
  • Anti-cancer-associated R ⁇ A EGS molecules can be designed by taking the basic structure of a pre- tR ⁇ A molecule (pre-tR ⁇ A 1 ⁇ ) and adding internal guide sequences, for example, by substituting the sequences of the aminoacyl acceptor stem and the D stem with sequences complementary to the PML-RAR ⁇ sequence around the fusion junction. Similar EGS molecules can be engineered for breakpoints having different sequences. EGS molecules can be readily screened for the ability to promote preferential cleavage by RNaseP of a particular cancer- associated RNA using the assayed described in Yuan, Y. , Hwayng, E.S. and Altman, S., Proc . Natl . Acad. Sci . , USA, 89, 8006-8010, (1992) .
  • Antisense nucleotides are typically 15-30 nucleotides long and are usually DNA-based. They cause inhibition of translation of mRNA by binding to the RNA and causing a translational block and by ' directing endogenous RNaseH to cleave the RNA.
  • the sequences appropriate for inhibition of translation, or increased susceptibility to degradation by the endogenous RNaseH, are designed based on the sequences unique to the leukemia, as shown below in Example 4. Several modifications can be introduced to the antisense DNA molecule to improve its nuclease stability. Nuclease Resistant Anti-Cancer-associated mRNA
  • Anti-cancer-associated mRNA ribozymes, EGS molecules, or antisense oligonucleotides can be produced which have a decreased susceptibility to intracellular degradation.
  • one or more of the bases of a ribozyme or EGS RNA construct can be replaced by 2' methoxy ribonucleotides or phosphorothioate deoxyribonucleotides using available nucleic acid synthesis methods (see, for example, Offensperger et. al . , EMBO J.
  • cytosines that may be present in the sequence can be methylated, or an intercalating agent, such as an acridine derivative, can be covalently attached to a 5' terminal phosphate (for example, using a pentamethylene bridge) to reduce the susceptibility of a nucleic acid molecule to intracellular nucleases (see, for example, Maher et al . , Science, 245 : 725-730 (1989) ; Grigoriev et al . , J. Biol . Chem. , 267 : 3389-3395 (1992)) .
  • an intercalating agent such as an acridine derivative
  • nucleotide' s ribose moiety Another class of possibly useful chemical modifications expected to be useful is modification of the 2' OH group of a nucleotide' s ribose moiety, which has been shown to be critical for the activity of the various intracellular and extracellular nucleases.
  • Typical 2' modifications are the synthesis of 2'-0-Methyl oligonucleotides (Paolella et al . , EMBO J., 11:1913-1919, 1992) and 2'- fluoro and 2' -amino-oligonucleotides (Pieken, et al., Sciences, 253:314-317, 1991; Heidenreich and Eckstain, J. Biol.
  • nuclease-resistant ribozyme constructs are shown in Figures 7a and 7b in which all of the nucleotides, except core nucleotides (in italics in Figures 7a and 7b) critical for efficient cleavage activity, are replaced with either 2'-0-methyl ribonucleotides or phosphorothioate deoxyribonucleotides .
  • WO 95/23225 by Ribozyme Pharmaceuticals describes chemical modifications for increasing the stability of ribozymes, which can also be used in EGSs, such as the introduction of an alkyl group at the 5' -position of a nucleoside or nucleotide sugar.
  • 5' -C-alkylnucleotides can be present in enzymatic molecules or antisense oligonucleotides for increased stability.
  • An alkyl group refers to a saturated aliphatic hydrocarbon, including straight-chain, branch chain, and cyclic alkyl groups with preferably 1 to 12 carbons.
  • WO 95/23225 also describes 2'-deoxy-2'- alklynucleotides which may be present to enhance the stability of oligonucleotides. For example, an oligonucleotide having at the 2'-position on the sugar molecule an alkyl moiety present where the nucleotide is not essential for function will be more stable.
  • WO 95/23225 also describes the use of 3' and/or 5' -CF 2 -phosphonate substituted nucleotides that maintain or enhance the catalytic activity and/or nuclease resistance of an enzymatic or antisense molecule.
  • ribozymes described in WO 95/23225 Another useful method for stabilization of ribozymes described in WO 95/23225 is increasing the length of helix 2 of a hairpin ribozyme (with or without helix 5) .
  • improved efficiency results from increasing helix 2 from 4 base pairs to 6 base pairs. The extent to which such modifications affect the efficiency with which the modified ribozyme or
  • EGS molecule promotes ribozyme-mediated cleavage of cancer-associated RNA can readily be determined using the cleavage assay described above.
  • Phosphorothioate antisense oligonucleotides directed against the PML-RAR ⁇ mRNA fusion junction can be synthesized on an automated DNA synthesizer by published methods (Agarwal, S., et al . , Proc .
  • oligonucleotide is chemically modified to increase resistance to nucleases, for example, by modification of the phosphodiester bond to methylphosphonate or phosphorothioate, or the substitution of the 2' position of the ribose with a methoxy, 0-alkyl, amino or fluoro group.
  • some modifications to the bases have the potential to enhance the binding (increase Tm) of the oligonucleotide to the target RNA.
  • RNA-based antisense molecule can also be expressed in the leukemia cells using a viral-vector.
  • the antisense molecules which are directed to the fusion junction of the PML-RAR ⁇ mRNA of the APL cells can bind to the RNA and inhibit the translation of the PML-RAR ⁇ mRNA by blocking its translation but not through RnaseH induced-cleavage of the PML-RAR ⁇ mRNA.
  • RNA antisense molecules are useful for eliciting continous inhibition of the PML-RAR ⁇ mRNA translation.
  • Preferred vectors for introducing anti-cancer- associated RNA ribozymes or EGS molecules into mammalian cells include viral vectors, such as the retroviruses, which introduce DNA which encodes an anti-cancer-associated mRNA ribozyme or EGS molecule directly into the nucleus where the DNA is then transcribed to produce the encoded ribozyme or EGS molecule.
  • Defective retroviral vectors which incorporate their own RNA sequence in the form of DNA into the host chromosome, can be engineered to incorporate an anti-cancer-associated mRNA ribozyme or EGS into the cells of a host, where copies of the ribozyme or EGS will be made and released into the cytoplasm or are retained in the nucleus to interact with the target nucleotide sequences of the particular cancer-associated mRNA.
  • Bone marrow stem cells and hematopoietic cells are relatively easily removed and replaced from humans, and provide a self-regenerating population of cells for the propagation of transferred genes.
  • Such cells could be transfected in vi tro or in vivo with retrovirus-based vectors encoding anti-cancer- associated mRNA ribozymes or EGS molecules .
  • vi tro transfection of stem cells is performed, once the transfected cells begin producing the particular anti-leukemia mRNA ribozymes or EGS molecules, the cells can be added back to the patient to establish entire clonal populations of cells that are resistant to leukemia formation.
  • FIG. 8 An example of a vector used to clone and express DNA sequences encoding the IHRZl.18 and IHRZl.3 anti-APL ribozyme constructs is shown in Figure 8.
  • This vector includes: 1. A cloning site in which to insert a DNA sequence encoding a ribozyme or EGS molecule to be expressed.
  • a mammalian origin of replication which allows episomal (non-integrative) replication such as the origin of replication derived from the Epstein-Barr virus.
  • a promoter such as one derived from Rous sarcoma virus (RSV) , cytomegalovirus (CMV) , or the promoter of the mammalian U6 gene (an RNA polymerase III promoter) which directs transcription in mammalian cells of the inserted
  • RSV Rous sarcoma virus
  • CMV cytomegalovirus
  • RNA polymerase III promoter the promoter of the mammalian U6 gene which directs transcription in mammalian cells of the inserted
  • DNA sequence encoding the ribozyme or EGS-encoding construct to be expressed is expressed.
  • a mammalian selection marker such as neomycin or hygromycin resistance, which permits selection of mammalian cells that are transfected with the construct.
  • a bacterial antibiotic resistance marker such as neomycin or ampicillin resistance, which permits the selection of bacterial cells that are transformed with the plasmid vector.
  • Anti-cancer-associated mRNA ribozymes, EGS molecules or antisense oligonucleotides can be used directly in combination with a pharmaceutically acceptable carrier to form a pharmaceutical composition suited for treating the particular leukemia.
  • a ribozyme, EGS for RNase P, or an RNA antisense may be delivered via a vector containing a sequence which encodes and expresses the ribozyme or EGS molecule specific for the leukemia-related mRNA produced in the leukemia cells .
  • Direct delivery involves the insertion of pre- synthesized ribozymes or EGS molecules or antisense molecules into the target cells, usually with the help of lipid complexes (liposomes) to facilitate the crossing of the cell membrane and other molecules, such as antibodies or other small ligands, to maximize targeting.
  • lipid complexes liposomes
  • directly delivered ribozymes and EGS molecules may be chemically modified, making them nuclease-resistant, as described above. This delivery methodology allows a more precise monitoring of the therapeutic dose.
  • Vector-mediated delivery involves the infection of the target cells with a self- replicating or a non-replicating system, such as a modified viral vector or a plasmid, which produces a large amount of the ribozyme encoded in a sequence carried on the vector.
  • a self- replicating or a non-replicating system such as a modified viral vector or a plasmid, which produces a large amount of the ribozyme encoded in a sequence carried on the vector.
  • Targeting of the cells and the mechanism of entry may be provided by the virus, or, if a plasmid is being used, methods similar to the ones described for direct delivery of ribozymes can be used.
  • Vector-mediated delivery will produce a sustained amount of ribozyme, EGS molecules or antisense, it will be substantially cheaper and will require less frequent administration than a direct delivery such as intravenous injection of the ribozyme, EGS molecules or antisense oligonucleotides. Being part of the hem
  • the direct delivery method may be used during the acute critical stages of the disease, when relatively rapid removal of the maturation blockage is desired.
  • intravenous or subcutaneous injection is used to deliver ribozymes or EGS molecules antisense directly.
  • the oligonucleotides be delivered in a form which prevents degradation of the oligonucleotide before it reaches the intended target site.
  • the disease enters a quiescent stage, with undifferentiated stem cells carrying the t(15;17) translocation, it may be useful to treat patients with vector-delivered ribozyme or EGS, allowing a continuous removal of the fusion mRNA and preventing future relapses.
  • the pharmaceutical carrier specifically delivers the ribozyme or EGS to affected cells.
  • APL affects hematopoietic cells
  • a preferred pharmaceutical carrier delivers anti-APL ribozymes, EGS, or antisense molecules to hematopoietic cells and, most preferably, only to the subset of hematopoietic cells affected by APL, promyelocytes, myeloid cell lines. Delivery of Ribozymes, EGS or Antisense Oligonucleotides
  • a patient will have to be typed, and the molecular structure of the patient's characteristic PML-RAR ⁇ mRNA fusion junction will have to be determined prior to ribozyme therapy.
  • the vast majority of APL patients have either Jbcr! or Jbcr3- type junctions, which means that one of two ribozymes will have to be used.
  • Treatment of APL patients with ribozymes, EGS, or antisense molecules will be carried out in two phases. Phase I is designed to treat the acute phase of the disease and may be carried out in combination with other drugs, including conventional chemotherapy and retinoic acid.
  • Two methods of delivery may be employed, (1) delivery of synthetic ribozymes, EGS, or antisense molecules, or (2) delivery of plasmids expressing ribozymes, EGS, or antisense molecules in a transient fashion.
  • the method of choice will have to be determined in preclinical studies, and it is possible that they may be used in combination. Both of them can be efficiently delivered, for example, by using cationic liposome preparations.
  • antibodies conjugated to the liposomes may be used.
  • a phase II treatment for APL patients may be recommended for those who show minimal residual disease, in spite of clinical remission, following the first phase of the treatment.
  • This phase of the treatment will include an autologous bone marrow transplant, where the bone marrow cells are treated with a ribozyme or EGS-delivering vector, to generate clones of cells that will not express the fusion gene product due to constant production of specific anti-APL ribozyme, EGS, or antisense molecules.
  • Transfection of bone marrow cells may be useful in the treatment of APL to treat bone marrow of patients who show minimal residual disease by PCR testing even though they are clinically in remission.
  • the bone marrow transfected with ribozyme, EGS or antisense-producing plasmids or retroviral vectors is then used to repopulate the hematopoietic system of the patient in an autologous bone marrow transplant.
  • EGSs EGSs, ribozymes or ones similar to them can either chemically synthesized or expressed using a vector can be used for the treatment of APL patients. Two approaches can be taken for the treatment of these patients.
  • Part of the bone marrow from these patients can be removed and the cells treated with the appropriate ribozymes or EGS to kill all APL cells.
  • the remaining marrow cells can then be expanded in culture in the presence of growth factors and IL2 cytokines.
  • the patient can then be cytoablated using a combination of chemotherapy and sub-lethal radiation therapy to destroy all APL cells in the patient.
  • the treated and expanded marrow cells can then be re-introduced into the patient. These cells can then repopulate the patient's vascular system and form the basis for a therapy for APL.
  • ribozymes or EGSs if used in a chemically synthesized form, can be either directly administered through an intravenous route or other standard modes of intake to kill all APL cells in the patient.
  • the chemically synthesized or vector- expressed ribozymes or EGSs can be delivered using liposomal formulations.
  • microparticles include liposomes, virosomes, microspheres and microcapsules formed of synthetic and/or natural polymers. Methods for making microcapsules and microspheres are known to those skilled in the art and include solvent evaporation, solvent casting, spray drying and solvent extension. Examples of useful polymers which can be incorporated into various microparticles include polysaccharides, polyanhydrides, polyorthoesters, polyhydroxides and proteins and peptides.
  • Liposomes can be produced by standard methods such as those reported by Kim, et al . , Biochim. Biophys . Acta, 728, 339-348 (1983) ; Liu, D., et al . , Biochim . Biophys . Acta , 1104 , 95-101 (1992) ; and Lee, et al . , Biochim . Biophys . Acta . , 1103 ,
  • Ribozyme, EGS, antisense molecules or DNA encoding such molecules can be encapsulated within liposomes when the molecules are present during the preparation of the microparticles. Briefly, the lipids of choice, dissolved in an organic solvent, are mixed and dried onto the bottom of a glass tube under vacuum.
  • the lipid film is rehydrated using an aqueous buffered solution of the ribozymes, EGS molecules, DNA encoding ribozymes, EGS molecules or antisense to be encapsulated, and the resulting hydrated lipid vesicles or liposomes encapsulating the material can then be washed by centrifugation and can be filtered and stored at 4°C.
  • This method has been used to deliver nucleic acid molecules to the nucleus and cytoplasm of cells of the MOLT-3 leukemia cell line (Thierry, A.R. and Dritschilo, A., Nucl. Acids Res . , 20 : 5691-5698 (1992)) .
  • ribozymes, EGS, antisense molecules, or DNA encoding such molecules can be incorporated within microparticles, or bound to the outside of the microparticles, either ionically or covalently.
  • Cationic liposomes or microcapsules are microparticles that are particularly useful for delivering negatively charged compounds such as nucleic acid-based compounds, which can bind ionically to the positively charged outer surface of these liposomes.
  • Various cationic liposomes have previously been shown to be very effective at delivering nucleic acids or nucleic acid-protein complexes to cells both in vi tro and in vivo, as reported by Feigner, P.L. et al . , Proc . Natl . Acad. Sci . USA, 84 : 7413-7417 (1987); Feigner, P.L., Advanced Drug Delivery Reviews, 5 : 163-187 (1990) ; Clarenc, J.P.
  • Cationic liposomes or microcapsules can be prepared using mixtures including one or more lipids containing a cationic side group in a sufficient quantity such that the liposomes or microcapsules formed from the mixture possess a net positive charge which will ionically bind negatively charged compounds.
  • positively charged lipids examples include the aminolipid dioleoyl phosphatidyl ethanolamine (PE) , which possesses a positively charged primary amino head group; phosphatidylcholine (PC) , which possess positively charged head groups that are not primary amines; and N[1- (2, 3-dioleyloxy)propyl] -N,N,N- triethylammonium ( "DOTMA, " see Feigner, P.L. et al., Proc . Natl . Acad. Sci USA, 84 , 7413-7417 (1987) ; Feigner, P.L. et al.
  • PE aminolipid dioleoyl phosphatidyl ethanolamine
  • PC phosphatidylcholine
  • DOTMA see Feigner, P.L. et al., Proc . Natl . Acad. Sci USA, 84 , 7413-7417 (1987) ; Feigner, P
  • Nucleic acid can also be encapsulated by or coated on cationic liposomes which can be injected intravenously into a mammal .
  • This system has been used to introduce DNA into the cells of multiple tissues of adult mice, including endothelium and bone marrow, where hematopoietic cells reside (see, for example, Zhu et al . , Science, 261 : 209-211 (1993) ) .
  • Liposomes containing either ribozymes, EGS, antisense molecules or DNA encoding these molecules can be administered systemically, for example, by intravenous or intraperitoneal administration, in an amount effective for delivery of the anti-cancer-associated mRNA ribozyme, EGS or antisense to targeted cells.
  • Other possible routes include trans-dermal or oral, when used in conjunction with appropriate microparticles.
  • the total amount of the liposome- associated nucleic acid administered to an individual will be less than the amount of the unassociated nucleic acid that must be administered for the same desired or intended effect.
  • compositions including various polymers such as the polylactic acid and polyglycolic acid copolymers, polyethylene, and polyorthoesters and the anti-APL ribozymes, EGS, antisense molecules, or DNA encoding such molecules, can be delivered locally to the appropriate cells by using a catheter or syringe.
  • Other means of delivering such compositions locally to cells include using infusion pumps (for example, from Alza Corporation, Palo Alto, California) or incorporating the compositions into polymeric implants (see, for example, P. Johnson and J.G.
  • Plasmids Plasmid pAPL 7-5, diagrammed in
  • Figure 8 was constructed by cloning a 788 nucleotide fragment spanning the PML-RAR ⁇ fusion region (nt 1060 to 1848 of SEQ ID NO. 1, corresponding to a PML sequence of nucleotides 1076-1739 of clone B16 and a RAR ⁇ sequence of nucleotides 1766-1890 of PML-RAR ⁇ clone B467 of de The, et al . , Cell , 66 : 675-684 (1991)) in the vector pCRlOOO (Invitrogen Corp., San Diego, CA) .
  • pCRlOOO Invitrogen Corp., San Diego, CA
  • This fragment was PCR amplified from total mRNA of a cell line whose breakpoint and sequence are identical to that of the NB4 cell line (de The, et al . , Cell , 66 : 675-684 (1991) , Lanotte, M. et al . , Blood, 77 : 1080-1086 (1991)) .
  • the sequence in the fusion region was verified to be identical to that previously reported (de The, et al . , Cell , 66 : 675- 684 (1991) ) .
  • Plasmid pRAR5 was constructed by cloning an J57coRI fragment of a plasmid containing the full length RAR ⁇ sequence from an M13 sequencing vector into the EcoRI cloning site of pGEMTM-3Z (Promega, Madison, Wisconsin) .
  • the desired construct had the RAR ⁇ coding sequence (see SEQ ID NO. 2) cloned downstream of the T7 promoter and was selected by restriction analysis. For transcription, this plasmid was linearized with AccI .
  • Oligonucleotides All the oligonucleotides used for transcription and sequencing were synthesized on an Applied Biosystems, Inc. (ABI) DNA synthesizer model 392. Gel-purified oligodeoxyribonucleotides were resuspended in 10 mM Tris HCI, pH 8.0, 1 mM EDTA and stored at -20°C. Ribozymes and EGS molecules were synthesized in vi tro by transcription with T7 RNA polymerase .
  • Hindlll-linearized pAPL-3Z3 generated a transcript containing 788 nucleotides of PML-RAR ⁇ and approximately 60 nucleotides of vector sequences at the 3' end while transcription of Accl-linearized pRAR5 generated a 960 nucleotide transcript. Transcription from oligonucleotides was carried out using a standard method essentially as described by Milligan, et al . ⁇ Nucl . Acids Res . , 15 : 8783-8798 (1987)) , using a complete coding strand and a partial complementary strand spanning the promoter region.
  • the plasmid expressing the RAR ⁇ sequence When linearized with the restriction enzyme AccI, the plasmid expressing the RAR ⁇ sequence generates a 960 nt transcript, corresponding to nt 1 to 960 of SEQ ID NO. 4. The site where the PML-RAR ⁇ fusion occurs in the recombined gene is located at nt 146 of this sequence.
  • RNAs were resuspended in water and stored at -20°C.
  • Ribozyme Cleavage Assay Cleavage reactions were carried out in a volume of 10 ⁇ l in 50 mM Tris HCI, pH 7.5, 30 mM MgCl 2 , for 3 hours at 37°C.
  • the reactions contained 0.03 ⁇ M of the radiolabeled RNA substrates and varying amounts of ribozyme (0,0.03, 0.1, 0.3, 1, 3 and 6 ⁇ M) .
  • the reactions were stopped by adding 10 ⁇ l of a stop solution (formamide containing 30 mM EDTA and tracing dyes 0.025% bromophenol blue and 0.025% xylene cyanol) , followed by heating at 90°C ' for 3 minutes.
  • a stop solution formamide containing 30 mM EDTA and tracing dyes 0.025% bromophenol blue and 0.025% xylene cyanol
  • Anti-APL ribozymes were synthesized by in vitro transcription using T7 RNA polymerase. Ribozymes were designed and synthesized to preferentially bind APL-associated PML-RAR ⁇ mRNA sequences associated with APL. There are two hammerhead ribozyme cleavage sites in the vicinity of the bcrl fusion junction: cleavage site 1 is an AUU located two nucleotides 3' to the PML-RAR ⁇ fusion sequence (nt 1727 to 1729 of Sequence ID No. 3) and cleavage site 2 is a UUC located twenty-two nucleotides 3' to the fusion (nt 1747 to 1749 of SEQ ID NO. 3) . Both of these cleavage sites are actually located in the RAR ⁇ portion of the PML- RAR ⁇ sequence .
  • Ribozyme construct IHRZl.18 was designed to target cleavage site 1 of the PML-RAR ⁇ fusion sequence.
  • the guide sequence (nt 29 to 36 SEQ ID NO. 5) of one of the arms of IHRZl.18 is complementary to an eight nucleotide sequence at and across the junction of the PML-RAR ⁇ fusion (nt 1721 to 1728 of SEQ ID No. 3) and forms helix III upon binding to the PML-RAR ⁇ substrate RNA molecule (SEQ ID NO. 3) (see Figure la) .
  • Ribozyme Construct IHRZl.3 Ribozyme construct IHRZl.3 (SEQ ID NO. 6) was designed to target cleavage site 2 of the PML-RAR ⁇ fusion sequence.
  • a portion of the guide sequence (nt 34 to 41 of SEQ ID NO. 6) of one arm of IHRZl .3 is complementary to and binds to an eight nucleotide sequence (nt 1721 to 1728 of SEQ ID NO. 3) at and across the junction of the PML-RAR ⁇ fusion RNA sequence ( Figure 2a) .
  • the same guide sequence is also complementary to a three nucleotide sequence 5' of cleavage site 2 and present in both the PML- RAR ⁇ and the RAR ⁇ substrate RNA molecules ( Figures 2a and 2b) .
  • Binding of this guide sequence to PML- RAR ⁇ substrate RNA results in a helix III interrupted by a looping out of seventeen nucleotides (nt 1729 to 1745 of SEQ ID NO. 3) of the PML-RAR ⁇ substrate RNA opposite a three nucleotide loop out of the IHRZl.3 construct ( Figure 2a) .
  • the portion of the interrupted helix III 5' of the seventeen nucleotide loop out is designated helix Illb and consists of the eight base pairs found in helix III of IHRZl.18 ( Figure la) .
  • helix III 3' from the seventeen nucleotide loop out of PML- RAR ⁇ substrate RNA is designated helix Ilia and consists of the above-mentioned three base pairs 5' of cleavage site 2. Binding of this guide sequence in IHRZl.3 to wild-type RAR ⁇ substrate RNA also results in an interrupted helix III (helices Ilia and Illb) in which helix Illb is formed by only four, instead of eight, base pairs 5' of the seventeen nucleotide loop out of the RAR ⁇ substrate RNA ( Figure 2b) .
  • Helix 1 of the ribozyme-substrate complex is formed between another arm of IHRZl.3 consisting of a five nucleotide guide sequence (nt 1 to nt 5 of SEQ ID NO. 6) and a sequence of the substrate RNA
  • Ribozyme construct IHRZl.30 (SEQ ID NO. 7) is designed to target cleavage site 1 of the PML-RAR ⁇ fusion sequence.
  • One portion of the guide sequence (nt 34 to 41 of SEQ ID NO. 7) of one arm of IHRZl.30 is complementary to eight nucleotides 5' of the PML- RAR ⁇ fusion junction (nt 1701 to 1708 of Sequence ID No. 3) and another portion of the same guide sequence is complementary to three nucleotides 5' of cleavage site 2 (3' to the fusion junction) (see Figure 3a) .
  • Binding of this guide sequence to the PML-RAR ⁇ substrate RNA forms a helix III interrupted by a looping out of 17 nucleotides (nt 1709 to 1725 of Sequence ID No. 3) of the PML-RAR ⁇ substrate RNA opposite a three nucleotide loop out of the IHRZl.30 sequence.
  • Helix Illb is formed by the complementary base pairing between the eight bases of the guide sequence and of the PML-RAR ⁇ sequence 5' of the 17 nucleotide loop out
  • helix Ilia is formed by the base pairing between three other nucleotides of the guide sequence and those of the PML-RAR ⁇ sequence 3' of the 17 nucleotide loop out (see Figure 3a) .
  • the complex that IHRZl.30 can form with RAR ⁇ is different from the one formed by PML-RAR ⁇ , as shown in Figure 3b.
  • This complex results in the formation of a stem between nt 35 to 38 of SEQ ID NO. 7 and nt 139-142 of SEQ ID NO. 2 (helix Illb) and a stem between nt 29 to 31 of SEQ ID NO. 7 and nt 147 to 149 of SEQ ID NO. 4 (helix Ilia) .
  • Helix Illb helix Illb
  • helix Ilia helix Ilia
  • Example 3 Screening for Anti-APL-Associated RNA Ribozyme Activity.
  • Example 2 The ribozyme constructs described in Example 2 were assayed using the standard cleavage assay described in Example 1 to determine the efficiency of the cleavage reaction against a PML-RAR ⁇ substrate RNA and against an RAR ⁇ substrate RNA.
  • IHRZl.18 efficiently cleaved both the PML-RAR ⁇ RNA and the RAR ⁇ substrate RNA molecules (Table I) .
  • IHRZl.18 is quite selective for the PML-RAR ⁇ fusion RNA, but this selectivity tapers off at higher concentrations of the ribozyme ( Figure 4) .
  • ribozyme IHRZl.3 displayed approximately four-fold lower activity towards site 2 of the PML-RAR ⁇ fusion RNA substrate than did IHRZl.18 (a k cat /K, sou of 164 M "1 s "1 for IHRZl .3 compared with 560 M "1 s '1 for IHRZl.18 in Table I.
  • IHRZl.3 appeared to be much more specific for the PML-RAR ⁇ RNA than the RAR ⁇ transcript for which it displayed a k cat /K,_ of only 2.3 M "1 s "1 (see Table I) .
  • Ribozyme construct IHRZl.30 a modified ribozyme modeled on the design of IHRZl .3, except directed to site 1 on the PML-RAR ⁇ RNA, actually cleaved the wild-type RAR ⁇ substrate RNA two to three-fold better than the PML-RAR ⁇ substrate RNA ( Figure 6 and Table I) .
  • Construct IHRZl.30 turned out to be a very weak ribozyme, and, as expected, it actually cleaved the RAR ⁇ mRNA better than the PML-RAR ⁇ mRNA ( Figure 6 and Table I) .
  • the ratio of the efficiency of cleavage of PML-RAR ⁇ substrate RNA sequence and of RAR ⁇ substrate RNA sequence by the IHRZl.30 ribozyme was extremely low (0.38, Table I) , reflecting this ribozyme's greater efficiency at cleaving the wild-type RAR ⁇ substrate RNA than the PML-RAR ⁇ substrate RNA.
  • IHRZl.18 and IHRZl .3 complement each other in their selectivity behavior, in different concentration ranges ( Figures 4 and 5) . With regard to their therapeutic use, it would be preferable to use the most active ribozyme, at concentrations where the best selectivity can be achieved.
  • ribozyme-dependent inactivation studies have shown, high doses are often required to inactivate mRNA molecules, and ribozyme synthesis must be driven by powerful promoters, as described above.
  • a ribozyme such as IHRZl.3, which displays selectivity at high concentrations, may be a better choice.
  • Anti-APL phosphorothioate antisense molecules complementary to the fusion junction of the PML- RAR ⁇ mRNA were synthesized on an automated oligonucleotide synthesizer by QCB Inc., Hopkinton, MA. These oligonucleotides were deprotected and desalted on a G25 gel filtration column. Four oligonucleotides sequences were synthesized.
  • Oligonucleotide As-APL 1 hybridizes to nucleotides 1713-1737 of PML-RAR ⁇ SEQ ID 1; As-APL2 hybridizes to nucleotides 1710-1734 of SEQ ID 1 and As-APL3 hybridizes to nucleotides 1716-1740 of SEQ ID 1.
  • These molecules when introduced into cells expressing the PML-RAR ⁇ mRNA should inhibit the production of the PML-RAR ⁇ hybrid protein and can thus relieve the maturation block of the APL cells and cause them to differentiate from the promyelocyte to the granulocyte.
  • These phosphorothioate molecules can either be delivered directly to APL cells in culture or can be complexed with cationic lipids and then delivered to the cells.
  • APL 1.0 had 16 nucleotide hybridizing arms while APL 1.1 had 29 nucleotide hybridizing arms and both ribozymes were designed to cleave the same site.
  • APL 5.0 was analogous to APL 1.0 except that it had a two nucleotide deletion in the catalytic core of the ribozyme thereby making it catalytically inactive ( Figure 9) .
  • Another series of cDNAs encoding ribozymes based on IDRZ 1.3 ( Figure 2a) named APL 2.0, 2.1, 6.0 and 6.1 ( Figure 10) were also synthesized and cloned into Eboplpp vector.
  • APL 2.0 had a 16 nucleotide hybridizing arms while APL 2.1 had 30 nucleotide hybridizing arms.
  • APL 6.0 and 6.1 were analogous to 2.0 and 2.1 except that it had a two nucleotide deletion in the catalytic core of the ribozyme thereby making it catalytically inactive ( Figure 10) .
  • All APL ribozymes constructs with the flanking self-cleaving ribozymes were cloned into a pGEM vector (Promega Corp.
  • the ribozymes bearing Eboplpp vectors, APL 1.0, APL 1.1, APL 2.0, APL 2.1 (SEQ ID NO. 14) , and the controls APL 5 and APL 6.1 were transfected into NB4 cells, a human APL cell line (M. Lanotte, et. al., Blood 77, 1080 (1991)) using electroporation techniques (Mossar M.M. , et. al., Oncogene, 9, 833 (1994)) .
  • the transfected cells were selected under low hygromycin (65 ⁇ g ml) .
  • the episomal copy number is expected to increase (Mossar M.M., et . al . , Oncogene, 9, 833 (1994)) thereby increasing the expression of the various ribozymes in cells.
  • the hygromycin dose was increased to 500 ⁇ g/ml culture media in order to increase the expression of the ribozymes.
  • the proliferative potential of the cells were assayed by the MTT proliferation assay (Mosmann T et . al . , Journal of Immunological Methods , 65, 55 (1983)) .
  • EGSs APL A 20 SEQ ID NO. 15
  • APL 1009 SEQ ID NO. 16 targeted to the fusion junction of PML RAR were chemically synthesized on an Applied Biosystems 394 DNA/RNA synthesizer.
  • the sequence of these EGSs and their chemical composition are shown in Figures 13a and 13c.
  • EGS A20D which lacked two nucleotide in the sequences corresponding to the T-loop of the EGS but was otherwise similar to A20 is shown in Figure 13b.
  • EGS APL 1017 shown in Figure 13d, lacked three nucleotides in the T-loop but was otherwise similar to APL 1009.
  • the control EGSs (A20D and APL 1017) were incapable of inducing cleavage of APL mRNA in presence of RNaseP and but could hybridize to the fusion junction.
  • the EGSs were purified by Reverse-phase HPLC, concentrated, and suspended in 2M NaCl to convert the EGS into the Na form and dialyzed extensively against water and then lyophilized.
  • the EGSs were suspended in water for test tube cleavage assay or in 150 mM NaCl for cell culture testing.
  • Test tube cleavage assay 3 ng of linearized pAPL 7-5 plasmid with HindlLI restriction enzyme was transcribed as described in Example 1 in presence of 32 P-ATP for 30 min. 0.25 ⁇ M (final concentration) of EGS and 2 ⁇ l of a purified preparation of RNase P from HeLa cells (Bartkiewicz, M. et. al., Genes and
  • APPLICANT Innovir Laboratories, Inc.
  • TITLE OF INVENTION Ribozyme-Mediated Inactivation of Leukemia-Associated RNA
  • CTCCCCTTCA GCTTCTCTTC ACGCACTCCA AGATCTAAAC CGAGAATCGA AACTAAGCTG 60
  • CAGGAGCCCA CCATGCCTCC CCCCGAGACC CCCTCTGAAG GCCGCCAGCC CAGCCCCAGC 180
  • CCCAGCCCTA CAGAGCGAGC CCCCGCTTCG GAGGAGGAGT TCCAGTTTCT GCGCTGCCAG 240
  • CCCCAGCCTC AGCCCCAGCT CCAACAGAAG CAGCCCGGCC ACCCACTCCC CGTGACCGCC 2940
  • GUGCCCAGCC CUCCCUCGCC ACCCCCUCUA CCCCGCAUCU ACAAGCCUUG CUUUGUCUGU 240 CAGGACAAGU CCUCAGGCUA CCACUAUGGG GUCAGCGCCU GUGAGGGCUG CAAGGGCUUC 300

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Abstract

RNA molecules, including ribozymes, external guide sequences for RNAse P, and antisense oligonucleotides have been constructed which promote ribozyme cleavage of, or block transcription of, respectively, specific cancer-associated RNA, for example, acute promyeloleukocytic leukemia-associated RNA, follicular lymphoma-associated RNA and chronic myelocytic leukemia-associated RNA. Methods of producing and using such RNA molecules are also described.

Description

RIBOZYME-MEDIATED INACTIVATION OF LEUKEMIA- ASSOCIATED RNA
Background of the Invention
This application is directed to methods and ribozyme and antisense oligonucleotides compositions designed to inactivate RNA molecules associated with malignancies arising from chromosomal translocations, especially specific leukemias, such as Acute Promyelocytic Leukemia (APL) .
Acute Promyelocytic Leukemia About 10% of acute myeloblastic leukemias
(A ) in adults is acute promyelocytic leukemia (APL, French American British Classification (FAB) M3), see arrell, R.P., et al . , New England J. Med. , 329, 177-189 (1993) for reviews) . The disease typically presents with a bleeding diathesis which is often exacerbated by chemotherapy, leading to a high rate of early mortality, primarily from intracranial hemorrhage. The bleeding diathesis is due to the presence of malignant promyelocytes which release procoagulant substances. These, in turn, activate the coagulation cascade, depleting fibrinogen, clotting factors and platelets.
While conventional chemotherapy can achieve complete remission in most patients, the five year survival averages only 35-45 percent. These figures do not include the high degree of early mortality (Warrell, R.P., et al . , New England J. Med . , 329, 177-189 (1993)) . A second avenue of therapy for APL patients involves the use of retinoids, in particular all- trans retinoic acid (ATRA; commercially available as tretinoin™, Hoffman La Roche, Nutley, NJ) . In several published studies tretinoin™ has been able to induce remission in about 48% of the patients treated (Warrell, R.P., et al . , New England J. Med. , 329, 177-189 (1993) ; Huang, M.E., et al . , Blood, 72, 567-572 (1988) ; Castaigne, S., et al . , Blood, 76, 1704-1709 (1990) ; Warrell, R.P., Jr., et al . , New Engl . J. Med. , 324, 1385-1393 (1991) ; Cheson, B.D., New England J. Med. , 327, 422-424 (1992)) . However, the duration of the remission is short, averaging 3.5 months, following which patients display an acquired resistance to the retinoid. This resistance is probably explained by an increased clearance of the drug from the bloodstream, due to the induction of cytochrome P- 450 enzymes and increased expression of cellular retinoic acid-binding proteins. Combination of retinoid treatment with conventional chemotherapy is actively pursued at present, with initial results indicating a 60 to 70% cure (Cheson, B.D., New England J. Med. , 327, 422-424 (1992)) . APL is consistently associated with a non- random chromosomal abnormality, characterized by a balanced and reciprocal translocation between the long arms of chromosomes 15 and 17 (t(15;17)) , found in over 90% of patient-derived APL cells (Kakizuka, A., et al . , Cell , 66, 663-674, (1991) ; de The, H., et al . , Cell , 66, 675-684 (1991) ; Pandolfi, P.P., et al., Oncogene, 6, 1285-1292 (1991) ; Chang, K.S., et al . , Mol . Cell . Biol . , 12, 800-810, (1992)) . This translocation results in a fusion between the retinoic acid receptor gene (RARα) and a gene for a putative transcription factor, PML. The fusion product, PML-RARα, displays altered transactivating properties compared with wildtype RARα gene product, which acts as a transcription enhancer in response to retinoic acid (RA) (Kakizuka, A., et al., Cell , 66, 663-674, (1991) ; de The, H., et al . , Cell , 66 , 675- 684 (1991); Pandolfi, P.P., et al . , Oncogene, 6, 1285-1292 (1991)) . It has been shown that ATRA induces maturation of the leukemia cells both in vivo (Warrell, R.P., et al . , New England J. Med . , 329, 177-189, (1991)) and in cultured cells (Lanotte, M. , et al., Blood, 77, 1080-1086, (1991)), explaining the clinical effect of retinoids. This retinoic acid (RA) -responsiveness is tightly linked to the presence of the PML-RARα gene product (Lanotte, M., et al . , Blood, 77, 1080- 1086, (1991) ; Miller, W.H. , Jr., et al . , Proc . Natl . Acad. Sci . USA, 89, 2694-2698 (1992)) . From these and other findings (Grignani, F., et al . , Cell , 74, 423-431, (1993)) , it is postulated that
PML-RARα functions as a dominant negative mutation, its product blocking myeloid differentiation. Evidence for the involvement of the PML-RARα protein in the pathogenesis of APL is provided by its expression in U937 cells, which results in a block in differentiation, increased sensitivity to RA, and increased cell survival in the presence of limiting serum in the culture media (Grignani, F., et al . , Cell , 74, 423-431, (1993)) . Molecular Characterization of APL
Virtually all the APL patients display immature promyelocytes with the previously mentioned t(15;17) translocation. The precise location of this translocation at the molecular level is important, because different sequences are generated at the fusion junctions. Studies of a series of APL patients have shown that there is a large degree of heterogeneity among the various PML-RARα transcripts (Miller, W. H., Jr., et al . , Proc . Na tl . Acad. Sci USA, 89, 2694-2698 (1992) ; Pandolfi, P.P., et al., EMBO J. , 11, 1397-1407 (1992) ) . There are three sources of variability: (1) alternative splicing on the PML side of the mRNA, (2) alternative polyadenylation sites on the PML-RARα side (3' end of the transcript) and (3) variable fusion points. Studies of a large number of APL cases have shown that the breakpoint in chromosome 17 is always located inside intron 2 of the RARα sequence (Miller, W. H., Jr., et al . , Proc . Na tl . Acad . Sci USA, 89, 2694-2698 (1992) ; Pandolfi, P.P., et al . , EMBO J. , 11, 1397-1407
(1992)) . This results in the presence of the same RARα sequence in all the variants of PML-RARα transcripts. Breakpoints in chromosome 15, on the PML gene are instead clustered in three different regions, defined as bcrl , bcr2 and bcr3 (Pandolfi, P.P., et al., EMBO J. , 11, 1397-1407 (1992)) . The Jbcrl region spans the whole length of intron 6 of the PML gene, and translocations involving this breakpoint result in the generation of a mature mRNA in which exon 6 of PML and exon 3 of RARα are spliced together. The bcr2 region spans a region encompassing a small portion of intron 4, exon 5, intron 5 and exon 6 of PML. Translocations involving this breakpoint are essentially different from one another and many of them occur inside PML exons, causing a large variation in the fusion sequences and, occasionally, generating aberrant reading frames, which code for aberrant and truncated proteins. The J cr3 region is located in intron 3 of PML and invariably results in a mRNA in which exon 3 of PML and exon 3 of RARα are spliced together. The sequence in the fusion junction is identical in all the J cr3 cases. Taken together, bcrl and Jbcr3-type junctions account for at least 80 percent of the tested APL cases (Pandolfi, P.P., et al . , EMBO J. , 11, 1397-1407 (1992)) , with one study finding Jbcrl-type junctions at twice the rate of J cr3-type ones (Miller, W.H., Jr., et al . , Proc . Na tl . Acad. Sci USA, 89, 2694-2698 (1992)) . Other Translocational Cancers Many other cancers have been reported in the literature as arising due to, or associated with, chromosomal translocations. Examples include RBTN2 and t[ll; 14] [pl3 ; qll] in T cell acute leukemia and erythropoiesis, translin in lymphoid neoplasms, T[5;14] [q34;qll] in acute lymphoblastic leukemia, T14;18 chromosomal translocations in follicular lymphoma, Non-Hodgkin' s lymphoma, Hodgkin's disease; T18 translocations in human synovial sarcomas; Burkitt's lymphoma; t[ll; 22] [q24 ; ql2] translocation in Ewing sarcoma; t[3p; 6p] and t [12q; 17p] translocations in human small cell lung carcinomas; and t[15; 19] translocation in diseminated mediastinal carcinoma. In many of these cases, the transcription product of the fusion or the fusion itself represent targets for therapy, if a therapeutic agent could be designed which would selectively kill or inactivate those cells having the translocation.
It is, therefore, an object of the present invention to provide molecules and methods for treating patients or cells derived from patients having cancerous cells arising from or characterized by chromosomal translocations. It is a further object of the present invention to provide molecules and methods for treating patients or cells derived from leukemia patients that inactivate specific cancer-associated RNA, including APL-associated RNA produced in the affected blood cells of leukemia patients. Summary of the Invention
RNA molecules, such as ribozymes and external guide sequence (EGS) molecules for RNAse P, are engineered to promote efficient and specific ribozyme cleavage of mRNA associated with various types of cancers, especially leukemia-associated mRNA, including mRNA associated with APL. Antisense molecules are also designed which are directed against specific cancer-associated mRNA to promote inhibition of its expression. Engineered RNA molecules are designed and synthesized which contain specific nucleotide guide sequences which enable a ribozyme or external guide sequence for RNAse P to preferentially bind to and promote ribozyme-mediated cleavage of a specific cancer- associated RNA, or to block transcription. Examples demonstrate that ribozymes and EGS molecules for RNAse P have been constructed that bind to and promote ribozyme cleavage of leukemia- associated RNA in cells. Methods for the determination of the activity of a ribozyme or an EGS for the purpose of construct-screening, as well as methods for using and producing such RNA molecules, are also disclosed.
Brief Description of the Drawings
Figure 1 is a schematic of the proposed intra- and interstrand complementary base binding structures formed by ribozyme IHRZ1.18 (SEQ ID NO. 5) with (a) a PML-RARα substrate RNA molecule (nucleotide (nt) 1721 to 1754 of SEQ ID NO. 3) and (b) with a RARα substrate RNA molecule (corresponding to nt 142 to 175 of SEQ ID No. 4) . The substrate RNA nucleotide sequence is shown in boldface characters. An asterisk indicates the location of the nucleotide which corresponds to the site in RARα mRNA which becomes fused to PML mRNA nucleotides by transcription of PML-RARα gene fusions . The cleavage sites are indicated by arrows.
Figure 2 is a schematic of the proposed intra- and interstrand complementary base binding structures formed by ribozyme IHRZ1.3 (SEQ ID NO. 6) with (a) a PML-RARα and (b) a RARα substrate RNA molecules. The substrate RNA nucleotide sequence is shown in boldface characters. An asterisk indicates the location of the nucleotide which corresponds to the site in RARα mRNA which becomes fused to PML mRNA nucleotides by transcription of PML-RARα gene fusions. The cleavage sites are indicated by arrows.
Figure 3 is a schematic of the proposed intra- and interstrand complementary base binding structures formed by ribozyme IHRZ1.30 (SEQ ID NO. 7) with (a) a PML-RARα substrate molecule (nt 1701 to 1754 of SEQ ID NO. 3) and (b) a RARα substrate RNA (nt 136 to 175 of SEQ ID NO. 4) molecule.
Figure 4 is a graph of percent substrate RNA molecules not cleaved as a function of ribozyme concentration (μm) as determined by in vi tro cleavage assay of PML-RARα (closed circles) and RARα (open circles) mRNA substrate molecules by IHRZ1.18. Each point is an average of three experiments.
Figure 5 is a graph of percent substrate RNA molecule not cleaved as a function of ribozyme concentration (μm) as determined by in vi tro cleavage assay of PML-RARα (closed circles) and RARα (open circles) mRNA substrate molecules by IHRZl.3. Each point is an average of three experiments.
Figure 6 is a graph of percent substrate RNA molecule not cleaved as a function of ribozyme concentration (μm) as determined by in vi tro cleavage assay of PML-RARα (closed circles) and RARα (open circles) mRNA substrate molecules by IHRZl.30. Each point is an average of three experiments. Figure 7 is a schematic of a nuclease- resistant ribozyme designed to have all non-core nucleotides replaced with 2' 0-methyl ribonucleotides or phosphorothioate deoxyribonucleotides. Unmodified core sequence nucleotides shown in italics. (a) IHRZl.18-related nuclease-resistant ribozyme (SEQ ID NO. 5) . (b) IHRZl.3-related nuclease-resistant ribozyme (SEQ ID NO. 6) .
Figure 8 is a diagram of a ribozyme expression vector.
Figure 9 is the structure and sequence of anti-APL hammerhead ribozyme constructs (SEQ ID NO. 12) targeted to APL transcripts (nt. 1-41 of SEQ ID NO. 13) . The underlined nucleotides are found in the .1 series. Changes in the 5.n series
(inactive control) are indicated: changed to C in 5.0 and 5.1, deleted in 5.0 and 5.1.
Figure 10 is the structure and sequence of anti-APL hammerhead ribozyme constructs (SEQ ID NO. 14) targeted to APL transcripts (nt. 3-50 of SEQ ID NO. 13) . The underlined nucleotides are found in the .1 series. Changes in the 6.n series (inactive control) are indicated: changed to C in 6.0 and 6.1, deleted in 6.0 and 6.1. Figure 11 is a graph of the MTT assay for inhibition of cell growth, plotting optical density (i.e., cell number) over time (days) for cells exposed to APL 2.0, 65 μg/ml of hygromycin (dark squares) ; APL 2.0 500 μg/ml of hygromycin (open squares) ; APL 2.1 65 μg/ml of hygromycin (dark diamonds) ; APL 2.1 500 μg/ml of hygromycin (open diamonds) ; APL 5 65 μg/ml of hygromycin (dark triangles) ; APL 5 500 μg/ml of hygromycin (open triangles) .
Figure 12 is a graph of the MTT assay for inhibition of cell growth, plotting optical density (i.e., cell number) over time (days) for cells exposed to APL 2.1 in combination with various concentrations of hygromycin: 65 μg/ml of hygromycin (dark squares) ; 130 μg/ml of hygromycin (open squares) ; 195 μg/ml of hygromycin (dark diamonds) ; 260 μg/ml of hygromycin (open diamonds) ; 325 μg/ml of hygromycin (dark triangles) ; 490 μg/ml of hygromycin (open triangles) .
Figures 13a, 13b, 13c, and 13d are the structures and sequences of external guide sequences targeted to the fusion junction of PML RAR. Figure 13a is APL EGS A20 (APL RNA is nt. 7- 24 of SEQ ID NO. 13; EGS RNA is SEQ ID NO. 15) ; Figure 13b is the inactive control A20D (SEQ ID NO. 15 minus nt 22 and 23); Figure 13c is the APL EGS
1009 (APL RNA is nt. 6-22 of SEQ ID NO. 13; EGS RNA is SEQ ID NO. 16) ; Figure 13d is the inactive control 1017 (SEQ ID NO. 15 minus nt 14, 17, 18, 29) . Figures 14a and 14b are graphs of the MTT assay for inhibition of cell growth, plotting optical density (i.e., number of cells) over time (days) , for APL target EGS A20 (Figure 14a) and inactive control EGS (Figure 14b) at concentrations of 10 μM (dark square) , 9 μM (open square) , 8 μM (dark diamond) , 7 μM (open diamond) , 6 μM (dark triangle) , 5 μM (open triangle) , 4 μM (dark circle) , 3 μM (open circle) , 2 μM (X) , and 1 μM (*) .
Figures 15a and 15b are graphs of the MTT assay for inhibition of cell growth, plotting optical density (i.e., number of cells) over time (days) , for APL target EGS 1009 (Figure 15a) and inactive control EGS (Figure 15b) at concentrations of 10 μM (dark square) , 9 μM (open square) , 8 μM (dark diamond) , 7 μM (open diamond) , 6 μM (dark triangle) , 5 μM (open triangle) , 4 μM (dark circle) , 3 μM (open circle) , 2 μM (X) , and 1 μM (*) •
Detailed Description of the Invention
RNA molecules suitable for use in the treatment of cancers associated with chromosomal translocations have been designed. In the preferred embodiments described herein, the RNA molecules are ribozymes or external guide sequences specifically binding to and cleaving RNA in specific leukemias, such as those of APL. I. Determination of Specific Cancer-Associated RNA Sequence.
Based on the studies described herein using APL as a model, ribozymes, EGSs, or antisense oligonucleotides can be designed which block transcription of the translocated RNA. As discussed above, the presence and analysis of specific translocations has been made in a number of types of cancers. Oligonucleotides are designed based on the same principles for these cancers as for APL RNA. In APL patients, APL-associated mRNA molecules encoding proteins associated with APL are formed by transcription of aberrant gene fusions characteristically found in the particular type of APL. Accordingly, the presence of specific APL- associated mRNA must first be identified. Once specific APL-associated mRNA has been identified, some nucleotide sequence data of that mRNA must be obtained so that ribozymes or EGS molecules can be engineered to bind to one or more nucleotide sequences uniquely characteristic of the particular APL-associated mRNA.
As noted above, virtually all APL patients display immature promyelocytes which contain a particular type of t(15;17) chromosomal translocation. Thus, the leukemic promyelocytes of a patient with APL contain RNA transcripts of the specific PML-RARα chromosomal translocation characteristic of that patient's type of APL. Accordingly, the presence of cancer-associated mRNA must be identified in a patient, and its sequence determined in order to design and use the ribozymes, EGS molecules and antisense oligonucleotides described herein to preferentially cleave, and thereby inactivate, the specific cancer-associated mRNA. Diagnosis of APL can be made using histopathological data, cytogenetic data and polymerase chain reaction (PCR) analysis. The PCR data should yield the kind of breakpoint present at the PML-RARα junction. More than 80% of the APL cases have either a bcr-1 or a Jbcr-3-type junction. In these cases, there is no need to design a new ribozyme or EGS, but one of the constructs described herein can be used, either presynthesized or cloned into a vector. For the remaining cases, where the junction is a j cr-2-type, if a ribozyme or EGS molecule is not available, one can be designed and synthesized as described below.
The identification of the various characteristic Jbcr sequences makes the typing of a particular APL routine using standard methods. The characteristic Jbcr sequences of the various types of APL can be used as probes in standard hybridization blots to identify PML-RARα RNA transcripts or PML-RARα gene fusions, and the nucleotide sequence of such PML-RARα RNA or DNA molecules can be routinely determined by standard cloning and nucleic acid sequencing methods (see, for example, Sambrook et al., In Molecular Cloning: A Laboratory Manual, second edition: Vol. 1: 7.39- 7.87 (RNA hybridization and sequence analysis) ;
Vol. 2: 13.1-13.104 (DNA sequencing methods)) .
II. Designing and Constructing Ribozymes, EGS Molecules and Antisense Oligonucleotides Directed Against Cancer-associated mRNA. A. Background.
Ribozymes and External Guide Sequence Molecules
Ribonucleic acid (RNA) molecules can serve not only as carriers of genetic information, for example, genomic retroviral RNA and messenger RNA (mRNA) molecules and as structures essential for protein synthesis, for example, transfer RΝA (tRΝA) and ribosomal RΝA (rRΝA) molecules, but also as enzymes which specifically cleave nucleic acid molecules. Such catalytic RNA molecules are called ribozymes .
The discovery of catalytic RΝA, by Drs . Altman and Cech, who were awarded the Νobel prize in 1989, has generated much interst in commercial applications, particularly in therapeutics (Altman, S., Proc . Na tl . Acad . Sci . USA 90, 10898-10900 (1993) ; Symons R.H., Annu. Rev. Biochem. , 61, 641- 671 (1992) ; Rossi, J.J., et al . , Antisense Res . Dev. , 1, 285-288 (1991) ; Cech, T. , Annu. Rev. Biochem. , 59, 543-568, (1990)) . Several classes of catalytic RNAs (ribozymes) have been described. Self -Splicing Intron -Derived Ribozymes
One major class of ribozymes includes certain RNA sequences known as intervening sequences or introns. These sequences are removed in order to generate the final functional RNA. It was shown that many members of two classes of introns, groups I and II, ubiquitously found in lower eukaryotes, can excise themselves without the help of protein factors. This kind of ribozyme was discovered by Thomas Cech and colleagues, who have discussed some in vitro applications (see, in PCT/US887/03161, published as WO 88/04300 16 June 1988, see also, Cech, T. , Annu. Rev. Biochem. , 59, 543-568, (1990) ) .
RNase P A second class of ribozymes include the RNA portion of an enzyme, RNAse P, which is involved in the processing of transfer RNA (tRNA) , a common cellular component of the protein synthesis machinery. Bacterial RNase P includes two components, a protein (C5) and an RNA (Ml) . Sidney Altman and his coworkers demonstrated that the Ml RNA is capable of functioning just like the complete enzyme, showing that in Escherichia coli the RNA is essentially the catalytic component, (Guerrier-Takada, C. , et al . , Cell , 35, 849-857, (1983)) . In subsequent work, Dr. Altman and colleagues developed a method for converting virtually any RNA sequence into a substrate for bacterial RNase P by using an external guide sequence (EGS) , having at its 5' terminus at least seven nucleotides complementary to the nucleotides 3' to the cleavage site in the RNA to be cleaved and at its 5' terminus the nucleotides NCCA (N is any nucleotide) (Forster, A.C. and Altman, S., Science, 238, 407-409 (1990)) . Using similar principles, EGS/RNase P-directed cleavage of RNA has been developed for use in eukaryotic systems, (Yuan, Y., Hwang, E.S., and Altman, S., Proc . Natl . Acad . Sci . USA, 89, 8006-8010 (1992)) . These external guide sequences have more stringent requirements, however. The EGSs contain sequences which are complementary to the target RNA and which forms secondary and tertiary structure akin to portions of a tRNA molecule. A eukaryotic EGS must contain at least seven nucleotides which base pair with the target sequence 3' to the intended cleavage site to form a structure like the amino acyl acceptor stem, nucleotides which base pair to form a stem and loop structure similar to the T stem and loop, followed by at least three nucleotides that base pair with the target sequence to form a structure like the dihydroxyuracil stem. The EGS can be made more resistant to nuclease degradation by including chemically modified nucleotides or nucleotide linkages. The external guide sequence and the RNase P catalytic RNA can be used together as separate molecules. Alternatively, the two sequences can be combined into a single oligonucleotide molecule possessing both targeting and catalytic functions. Such a combined oligonucleotide, termed an RNase P internal guide sequence (RIGS) , increases the kinetic efficiency of cleavage by reducing the number of reactants and by keeping the targeting and catalytic elements in close proximity. Chemically modifying the nucleotides and phosphate linkages of EGS molecules and RIGS molecules make the oligonucleotides more resistant to nuclease degradation.
Viroid-Like Pathogens (VLP) -Derived Ribozymes
A third class of ribozymes is derived from the so-called viroid-like pathogens (VLP) , a group of self-replicating RNAs that include some plant pathogens such as viroids, virusoids and satellites of plant viruses and the hepatitis delta virus (HDV) , a human pathogen that functions as a satellite of hepatitis B virus. A key element of the life cycle of these pathogens is their replication strategy that involves synthesis of multimeric strands of both polarities (Branch, A.D. and Robertson, H.D., Science, 223, 450-455 (1988)) . These multimeric units are then cleaved into monomeric units by a self-cleavage activity, present in a specific region of the sequence. These sequences maintain their cleaving activity once they are separated from the bulk of the sequence and can also be engineered to cleave other sequences.
Most of the VLP-derived ribozymes known so far belong to the "hammerhead" subclass (Sy ons R.H., Annu. Rev. Biochem. , 61, 641-671 (1992) ; Forster, A.C, and Symons, R.H., Cell , 49, 211-220, (1987) ; Uhlenbeck, O . C . , Na ture, 328, 596-600 (1987) ; Haseloff, J., and Gerlach, W.L., Nature, 334, 585- 591 (1988)) . One ribozyme, derived from the antigenomic strand of the satellite of the tobacco ringspot virus, belongs to the "hairpin" subclass (Symons R.H., Annu. Rev. Biochem. , 61, 641-671 (1992) ; Hampel, A., Tritz, R., Biochemistry 28, 4929-4933 (1989) ) and two, derived from the genomic and the antigenomic stands of HDV are members of a third subclass, called "axehead" (Branch, A.D. and Robertson, Proc . Natl . Acad. Sci . USA, 88, 10163- 10167 (1991) ) .
Antisense Oligonucleotides
Antisense molecules are usually single stranded DNA or RNA molecules, or their substituted analogues, which can bind to the target RNA through Watson and Crick base pairing and prevent the translation of these RNAs (Mizuno, T. , et al., Proc. Na tl . Acad. Sci . USA, 81, (1983) ; Zamecnik, in Prospects for Antisense Nucleic Acid Therapy of Cancer and Aids, ed. , Wickstrom, Wiley-Liss, New York) ) . They are usually 15 to 30 nucleotides long and have been used widely to inhibit expression of various proteins (Zamecnick, P.C. and Stevenson, M.L. Proc . Natl . Acad . Sci . , USA, 75, 280 (1978) ;
Agrawal, S., Proc . Natl . Acad. Sci . , USA, 85, 7089, (1988)) . In addition to the inhibition of translation of the mRNA, DNA based antisense can also inhibit expression of proteins by presenting the DNA-RNA hybrid as a target for cleavage by the endogenous RNaseH enzyme (Giles, R.V. and Tidd, D.M., Nucleic Acid Res . , 20, 763 (1992)) , thereby destroying the target RNA. The antisense molecules can be made more resistant to nucleases by introducing phosphorothioate diester linkages instead of the phosphodiester linkage (Agrawal, S., et al . , Proc . Na tl . Acad . Sci . , USA, 85, 7089, (1988) ) and duplexes of these molecules with an RNA is recognized by RNaseH. B. Design and Synthesis of RNA Molecules.
Once the sequence of a particular cancer- associated RNA is determined, ribozymes and EGS molecules can be designed and synthesized which preferentially hybridize to the characteristic cancer-associated mRNA sequence and promote ribozyme-mediated cleavage or blockage of transcription or translation of that mRNA. For example, in APL, the leukemia cells contain mRNA transcripts of the PML-RARα gene fusion characteristic of that particular type of APL. The basic strategy for designing ribozymes or EGS molecules efffective against a particular type of APL is to engineer into ribozymes, EGS molecules, or antisense oligonucleotides, specific nucleotide guide sequences complementary to both sides of the particular PML-RARα gene fusion. Such engineered nucleotide guide sequences enable the ribozymes and EGS molecules to preferentially bind specific cancer-associated mRNA molecules and promote the subsequent ribozyme cleavage of, or block transcription of, the mRNA molecules.
The ability of an engineered ribozyme or EGS to promote ribozymal activity is readily determined using an in vi tro assay for a ribozyme's activity against a specific cancer-associated mRNA sequence, as described in more detail below. In the case of APL, the assay permits one to compare the efficiency of ribozymal cleavage against a particular PML-RARα mRNA sequence, characteristic of that type of APL, with an unaltered wild-type RARα mRNA sequence found in normal cells.
All the cells of the hematopoietic system express a functional retinoic acid receptor α (RARα) . This includes the immature promyelocytes characteristic of APL and their normal relatives. RARα is most likely essential for the survival and normal development of all the blood cells. Thus, it is general practice to make sure that enough RARα is being expressed to assure the patient's survival. Accordingly, a desired anti-cancer ribozyme or EGS molecule is one that preferentially promotes destruction of the PML-RARα mRNA, but not the RARα mRNA.
To demonstrate the feasibility of this strategy of designing and using ribozymes to specifically degrade cancer-associated mRNA, hammerhead ribozymes which cleave 3 ' of the target sequence NUH (N=A,U,G,C; H=A,U,C) were designed to bind RNA transcripts of particular PML-RARα gene fusions of APL. In order to test the ribozymes in vitro, plasmids encoding portions of the PML-RARα gene and the RARα gene, were synthesized. These plasmids allow the synthesis of shortened versions of APL mRNA molecules in vi tro, facilitating the testing and screening process. The mRNAs used in the examples described below contained the sequence of the PML-RARα fusion junction as it is found in NB4 cells. NB4 cells have been derived from APL patients (Lanotte, et al . , Blood, 77:1080 (1991)) . These cells possess a t(15;17) translocation {bcrl - type) , and they respond to treatment with ATRA, just as observed in cells of APL patients.
Methods to produce or synthesize ribozymes, EGS molecules, and DNA sequences encoding ribozymes or EGS molecules having a known sequence, are now routine using automated nucleic acid synthesis, for example, using the cyanoethyl phosphoramidite method on a DNA model 392 synthesizer by Applied Biosystems, Inc. (Foster City, CA) or a Pharmacia Oligo Pilot (Pharmacia, Piscataway, NJ) . Other methods for synthesizing nucleic acid molecules are also available (see, for example, Ikuta et al. , in Ann. Rev. Biochem. , 53 : 323-356 (1984) (phosphotriester and phosphite-triester methods) ; Narang et al . , in Methods Enzymol . , 65 : 610-620 (1980) (phosphotriester method) . Alternatively, ribozymes and EGS molecules can be synthesized by transcribing DNA templates, for example, with T7
RNA polymerase (Milligan, et al . , Nucl Acids Res . ,
15:8783 (1987) ) .
Anti-Cancer-Associated RNA Activity of Ribozyme Constructs
An in vi tro cleavage assay which measures the percentage of substrate RNA remaining after incubation with various amounts of an engineered ribozyme or EGS, in the presence of a non-limiting amount of RNAse P, is used as an indicator of the potential anti-leukemic activity of the ribozyme or the EGS/RNase P complex. Ribozymes or EGS/RNase P that exhibit the highest in vi tro activity are selected for further testing. The percentage of RNA remaining can be plotted as a function of the ribozyme (or EGS) concentration. The catalytic efficiency of a ribozyme can be expressed as k^/K,. (where kcat is the rate constant of cleavage and K„ is the Michaelis constant) , the second order rate constant for the reaction of a free ribozyme molecule (such as a hammerhead ribozyme) and substrate RNA molecule. Following the methods of Heidenreich and Eckstein (J". Biol . Chem . , 267 : 1904-1909 (1992)) ,
Figure imgf000021_0001
is determined using the formula
-In F/t = (k^/K,,, [C] where F is the fraction of substrate left, t is the reaction time, and [C] is the ribozyme concentration. Preferred ribozyme or EGS constructs are those which bind to and promote the preferential ribozyme cleavage of the cancer-associated substrate mRNA. Preferred constructs can be selected using the ribozyme cleavage assay, as shown by Example 1, and determining which constructs are the most efficient at specifically cleaving the cancer-associated substrate RNA sequence as determined by the value of
Figure imgf000022_0001
, as described above. Furthermore, when the cancer-associated mRNA contains sequences of a known wild-type mRNA found in normal cells, a more preferred ribozyme construct can be selected as the ribozyme which has the highest value of the ratio of the efficiency of ribozyme-mediated cleavage of the cancer-associated mRNA sequence and the efficiency of ribozyme cleavage of the related wild-type mRNA sequence found in normal cells, that is,
Figure imgf000022_0002
(cancer-associated RNA) -.
Figure imgf000022_0003
(wild-type RNA) . As explained above, this is the case for APL where the APL-associated PML-RARα mRNA shares sequences with the wild-type RARα mRNA . Thus, the more preferred ribozyme construct is one having the highest ratio of k^/i. (PML-RARα RΝA) :kcat/K,. (wildtype RARα RΝA) (see Example 3, below) .
Construction of an Anti-Leukemia EGS for RΝAse
P
Anti-cancer-associated RΝA EGS molecules can be designed by taking the basic structure of a pre- tRΝA molecule (pre-tRΝA1^) and adding internal guide sequences, for example, by substituting the sequences of the aminoacyl acceptor stem and the D stem with sequences complementary to the PML-RARα sequence around the fusion junction. Similar EGS molecules can be engineered for breakpoints having different sequences. EGS molecules can be readily screened for the ability to promote preferential cleavage by RNaseP of a particular cancer- associated RNA using the assayed described in Yuan, Y. , Hwayng, E.S. and Altman, S., Proc . Natl . Acad. Sci . , USA, 89, 8006-8010, (1992) .
Construction of Anti-Leukemia Antisense. Antisense nucleotides are typically 15-30 nucleotides long and are usually DNA-based. They cause inhibition of translation of mRNA by binding to the RNA and causing a translational block and by ' directing endogenous RNaseH to cleave the RNA. The sequences appropriate for inhibition of translation, or increased susceptibility to degradation by the endogenous RNaseH, are designed based on the sequences unique to the leukemia, as shown below in Example 4. Several modifications can be introduced to the antisense DNA molecule to improve its nuclease stability. Nuclease Resistant Anti-Cancer-associated mRNA
Constructs
Anti-cancer-associated mRNA ribozymes, EGS molecules, or antisense oligonucleotides can be produced which have a decreased susceptibility to intracellular degradation. For example, one or more of the bases of a ribozyme or EGS RNA construct can be replaced by 2' methoxy ribonucleotides or phosphorothioate deoxyribonucleotides using available nucleic acid synthesis methods (see, for example, Offensperger et. al . , EMBO J. , 12 : 1257-1262 (1993) ( in vivo use of antisense phosphorothioate oligodeoxynucleotides) ; PCT WO 93/01286 BY Rosenberg et al . , (synthesis of sulfurthioate oligonucleotides) ; Agrawal et al . , Proc . Na tl .
Acad. Sci . USA, 85 : 7079-7083 (1988) (synthesis of antisense oligonucleoside phosphoramidates and phosphorothioates to inhibit replication of human immunodeficiency virus-1) ; Sarin et al . , Proc . Na tl . Acad . Sci . USA, 85 : 7448-7794 (1989) (synthesis of antisense methylphosphonate oligonucleotides) ; Shaw et al . , Nucl eic Acids Res , 19 : 747-750 (1991) (synthesis of 3' exonuclease- resistant oligonucleotides containing 3' terminal phosphoroamidate modifications) ; incorporated herein by reference) . Another method expected to be useful to reduce susceptibility to 3' exonucleases is introduction of a free amine to a 3' terminal hydroxyl group of the ribozyme or EGS molecule (see, for example, Orson et al . , Nucl . Acids Res . , 19 : 3435-3441 (1991)) . Furthermore, cytosines that may be present in the sequence can be methylated, or an intercalating agent, such as an acridine derivative, can be covalently attached to a 5' terminal phosphate (for example, using a pentamethylene bridge) to reduce the susceptibility of a nucleic acid molecule to intracellular nucleases (see, for example, Maher et al . , Science, 245 : 725-730 (1989) ; Grigoriev et al . , J. Biol . Chem. , 267 : 3389-3395 (1992)) .
Another class of possibly useful chemical modifications expected to be useful is modification of the 2' OH group of a nucleotide' s ribose moiety, which has been shown to be critical for the activity of the various intracellular and extracellular nucleases. Typical 2' modifications are the synthesis of 2'-0-Methyl oligonucleotides (Paolella et al . , EMBO J., 11:1913-1919, 1992) and 2'- fluoro and 2' -amino-oligonucleotides (Pieken, et al., Sciences, 253:314-317, 1991; Heidenreich and Eckstain, J. Biol. Chem, 267:1904-1909, 1992) . Examples of suggested nuclease-resistant ribozyme constructs are shown in Figures 7a and 7b in which all of the nucleotides, except core nucleotides (in italics in Figures 7a and 7b) critical for efficient cleavage activity, are replaced with either 2'-0-methyl ribonucleotides or phosphorothioate deoxyribonucleotides .
WO 95/23225 by Ribozyme Pharmaceuticals describes chemical modifications for increasing the stability of ribozymes, which can also be used in EGSs, such as the introduction of an alkyl group at the 5' -position of a nucleoside or nucleotide sugar. 5' -C-alkylnucleotides can be present in enzymatic molecules or antisense oligonucleotides for increased stability. An alkyl group refers to a saturated aliphatic hydrocarbon, including straight-chain, branch chain, and cyclic alkyl groups with preferably 1 to 12 carbons. WO 95/23225 also describes 2'-deoxy-2'- alklynucleotides which may be present to enhance the stability of oligonucleotides. For example, an oligonucleotide having at the 2'-position on the sugar molecule an alkyl moiety present where the nucleotide is not essential for function will be more stable. WO 95/23225 also describes the use of 3' and/or 5' -CF2-phosphonate substituted nucleotides that maintain or enhance the catalytic activity and/or nuclease resistance of an enzymatic or antisense molecule. Another useful method for stabilization of ribozymes described in WO 95/23225 is increasing the length of helix 2 of a hairpin ribozyme (with or without helix 5) . For example, improved efficiency results from increasing helix 2 from 4 base pairs to 6 base pairs. The extent to which such modifications affect the efficiency with which the modified ribozyme or
EGS molecule promotes ribozyme-mediated cleavage of cancer-associated RNA can readily be determined using the cleavage assay described above. Nuclease-Resistant Antisense Oligonucleotides
Directed Against Leukemia mRNA
Phosphorothioate antisense oligonucleotides directed against the PML-RARα mRNA fusion junction can be synthesized on an automated DNA synthesizer by published methods (Agarwal, S., et al . , Proc .
Na tl . Acad . Sci . USA, 85, 7079 (1988) ) . The modifications described above, including wherein the oligonucleotide is chemically modified to increase resistance to nucleases, for example, by modification of the phosphodiester bond to methylphosphonate or phosphorothioate, or the substitution of the 2' position of the ribose with a methoxy, 0-alkyl, amino or fluoro group. In addition, some modifications to the bases have the potential to enhance the binding (increase Tm) of the oligonucleotide to the target RNA. One example of such a modification is the introduction of a propyne moiety to the C5 carbon of the nucleotide base, as described by Wagner, et al . , Science 260, 1510-1513 (1993) . A RNA-based antisense molecule can also be expressed in the leukemia cells using a viral-vector. The antisense molecules which are directed to the fusion junction of the PML-RARα mRNA of the APL cells can bind to the RNA and inhibit the translation of the PML-RARα mRNA by blocking its translation but not through RnaseH induced-cleavage of the PML-RARα mRNA. Such vector expressed RNA antisense molecules are useful for eliciting continous inhibition of the PML-RARα mRNA translation. Ill. Cloning and Expression Vectors Preferred vectors for introducing anti-cancer- associated RNA ribozymes or EGS molecules into mammalian cells include viral vectors, such as the retroviruses, which introduce DNA which encodes an anti-cancer-associated mRNA ribozyme or EGS molecule directly into the nucleus where the DNA is then transcribed to produce the encoded ribozyme or EGS molecule.
Examples of methods for using retroviral vectors for gene therapy are described in U.S. Patent Nos. 4,868,116 and 4,980,286; PCT applications PCT/US89/03794 and PCT/US89/00422; and Mulligan, Science, 260 : 926-932 (1993) ; the teachings of which are incorporated herein by reference.
Defective retroviral vectors, which incorporate their own RNA sequence in the form of DNA into the host chromosome, can be engineered to incorporate an anti-cancer-associated mRNA ribozyme or EGS into the cells of a host, where copies of the ribozyme or EGS will be made and released into the cytoplasm or are retained in the nucleus to interact with the target nucleotide sequences of the particular cancer-associated mRNA.
Bone marrow stem cells and hematopoietic cells are relatively easily removed and replaced from humans, and provide a self-regenerating population of cells for the propagation of transferred genes. Such cells could be transfected in vi tro or in vivo with retrovirus-based vectors encoding anti-cancer- associated mRNA ribozymes or EGS molecules . When in vi tro transfection of stem cells is performed, once the transfected cells begin producing the particular anti-leukemia mRNA ribozymes or EGS molecules, the cells can be added back to the patient to establish entire clonal populations of cells that are resistant to leukemia formation. An example of a vector used to clone and express DNA sequences encoding the IHRZl.18 and IHRZl.3 anti-APL ribozyme constructs is shown in Figure 8. This vector includes: 1. A cloning site in which to insert a DNA sequence encoding a ribozyme or EGS molecule to be expressed.
2. A mammalian origin of replication which allows episomal (non-integrative) replication, such as the origin of replication derived from the Epstein-Barr virus. 3. An origin of replication functional in bacterial cells for producing required quantities of the DNA encoding the ribozyme or EGS constructs, such as the origin of replication derived from the pBR322 plasmid.
4. A promoter, such as one derived from Rous sarcoma virus (RSV) , cytomegalovirus (CMV) , or the promoter of the mammalian U6 gene (an RNA polymerase III promoter) which directs transcription in mammalian cells of the inserted
DNA sequence encoding the ribozyme or EGS-encoding construct to be expressed.
5. A mammalian selection marker (optional), such as neomycin or hygromycin resistance, which permits selection of mammalian cells that are transfected with the construct.
6. A bacterial antibiotic resistance marker, such as neomycin or ampicillin resistance, which permits the selection of bacterial cells that are transformed with the plasmid vector.
IV . Therapy
Pharmaceutical Compositions
Anti-cancer-associated mRNA ribozymes, EGS molecules or antisense oligonucleotides can be used directly in combination with a pharmaceutically acceptable carrier to form a pharmaceutical composition suited for treating the particular leukemia. Alternatively, a ribozyme, EGS for RNase P, or an RNA antisense may be delivered via a vector containing a sequence which encodes and expresses the ribozyme or EGS molecule specific for the leukemia-related mRNA produced in the leukemia cells .
Direct delivery involves the insertion of pre- synthesized ribozymes or EGS molecules or antisense molecules into the target cells, usually with the help of lipid complexes (liposomes) to facilitate the crossing of the cell membrane and other molecules, such as antibodies or other small ligands, to maximize targeting. Because of the sensitivity of RNA to degradation, in many instances, directly delivered ribozymes and EGS molecules may be chemically modified, making them nuclease-resistant, as described above. This delivery methodology allows a more precise monitoring of the therapeutic dose.
Vector-mediated delivery involves the infection of the target cells with a self- replicating or a non-replicating system, such as a modified viral vector or a plasmid, which produces a large amount of the ribozyme encoded in a sequence carried on the vector. Targeting of the cells and the mechanism of entry may be provided by the virus, or, if a plasmid is being used, methods similar to the ones described for direct delivery of ribozymes can be used. Vector-mediated delivery will produce a sustained amount of ribozyme, EGS molecules or antisense, it will be substantially cheaper and will require less frequent administration than a direct delivery such as intravenous injection of the ribozyme, EGS molecules or antisense oligonucleotides. Being part of the hematopoietic system, malignant promyelocytes and their precursors are a prospective good target for retrovirus-derived vectors .
In the specific case of APL, the direct delivery method may be used during the acute critical stages of the disease, when relatively rapid removal of the maturation blockage is desired. Preferably, intravenous or subcutaneous injection is used to deliver ribozymes or EGS molecules antisense directly. It is essential that the oligonucleotides be delivered in a form which prevents degradation of the oligonucleotide before it reaches the intended target site. When the disease enters a quiescent stage, with undifferentiated stem cells carrying the t(15;17) translocation, it may be useful to treat patients with vector-delivered ribozyme or EGS, allowing a continuous removal of the fusion mRNA and preventing future relapses.
Most preferably, the pharmaceutical carrier specifically delivers the ribozyme or EGS to affected cells. For example, APL affects hematopoietic cells, and therefore, a preferred pharmaceutical carrier delivers anti-APL ribozymes, EGS, or antisense molecules to hematopoietic cells and, most preferably, only to the subset of hematopoietic cells affected by APL, promyelocytes, myeloid cell lines. Delivery of Ribozymes, EGS or Antisense Oligonucleotides
A patient will have to be typed, and the molecular structure of the patient's characteristic PML-RARα mRNA fusion junction will have to be determined prior to ribozyme therapy. The vast majority of APL patients have either Jbcr! or Jbcr3- type junctions, which means that one of two ribozymes will have to be used. Treatment of APL patients with ribozymes, EGS, or antisense molecules will be carried out in two phases. Phase I is designed to treat the acute phase of the disease and may be carried out in combination with other drugs, including conventional chemotherapy and retinoic acid.
Two methods of delivery may be employed, (1) delivery of synthetic ribozymes, EGS, or antisense molecules, or (2) delivery of plasmids expressing ribozymes, EGS, or antisense molecules in a transient fashion. The method of choice will have to be determined in preclinical studies, and it is possible that they may be used in combination. Both of them can be efficiently delivered, for example, by using cationic liposome preparations. In order to efficiently direct the synthetic ribozymes or EGS molecules, or retroviral vectors or plasmids encoding the ribozyme or EGS molecules, to the leukemic cells, antibodies conjugated to the liposomes may be used. These antibodies recognize surface antigens specific to the cell lineage that give rise to the APL cells. An active ribozyme, EGS, or antisense molecule in the APL cell will shut down the production of the rearranged fusion product. This will lead to the removal of the differentiation blockage seen in the APL cells and to the loss of the oncogenic effect of the rearranged gene. A phase II treatment for APL patients may be recommended for those who show minimal residual disease, in spite of clinical remission, following the first phase of the treatment. This phase of the treatment will include an autologous bone marrow transplant, where the bone marrow cells are treated with a ribozyme or EGS-delivering vector, to generate clones of cells that will not express the fusion gene product due to constant production of specific anti-APL ribozyme, EGS, or antisense molecules.
Transfection of bone marrow cells may be useful in the treatment of APL to treat bone marrow of patients who show minimal residual disease by PCR testing even though they are clinically in remission. The bone marrow transfected with ribozyme, EGS or antisense-producing plasmids or retroviral vectors is then used to repopulate the hematopoietic system of the patient in an autologous bone marrow transplant.
Methods of Delivering Anti-Cancer-associated mRNA Ribozymes or EGS Molecules to Cells
These EGSs, ribozymes or ones similar to them can either chemically synthesized or expressed using a vector can be used for the treatment of APL patients. Two approaches can be taken for the treatment of these patients.
1. Purging: Part of the bone marrow from these patients can be removed and the cells treated with the appropriate ribozymes or EGS to kill all APL cells. The remaining marrow cells can then be expanded in culture in the presence of growth factors and IL2 cytokines. The patient can then be cytoablated using a combination of chemotherapy and sub-lethal radiation therapy to destroy all APL cells in the patient. The treated and expanded marrow cells can then be re-introduced into the patient. These cells can then repopulate the patient's vascular system and form the basis for a therapy for APL.
2. Systemic administration: the ribozymes or EGSs if used in a chemically synthesized form, can be either directly administered through an intravenous route or other standard modes of intake to kill all APL cells in the patient.
Alternatively the chemically synthesized or vector- expressed ribozymes or EGSs can be delivered using liposomal formulations.
A variety of non-vector methods are available for delivering anti-cancer-associated mRNA ribozymes, EGS, or antisense molecules to leukemia cells. For example, in general, the anti-leukemia ribozymes, EGS, antisense molecules, or DNA sequences encoding the anti-leukemia ribozymes, EGS, or RNA antisense molecules, can be incorporated within or on microparticles . As used herein, microparticles include liposomes, virosomes, microspheres and microcapsules formed of synthetic and/or natural polymers. Methods for making microcapsules and microspheres are known to those skilled in the art and include solvent evaporation, solvent casting, spray drying and solvent extension. Examples of useful polymers which can be incorporated into various microparticles include polysaccharides, polyanhydrides, polyorthoesters, polyhydroxides and proteins and peptides.
Liposomes can be produced by standard methods such as those reported by Kim, et al . , Biochim. Biophys . Acta, 728, 339-348 (1983) ; Liu, D., et al . , Biochim . Biophys . Acta , 1104 , 95-101 (1992) ; and Lee, et al . , Biochim . Biophys . Acta . , 1103 ,
185-197 (1992) ; Wang, et al . , Biochem., 28, 9508- 9514 (1989)) , incorporated herein by reference. Ribozyme, EGS, antisense molecules or DNA encoding such molecules, can be encapsulated within liposomes when the molecules are present during the preparation of the microparticles. Briefly, the lipids of choice, dissolved in an organic solvent, are mixed and dried onto the bottom of a glass tube under vacuum. The lipid film is rehydrated using an aqueous buffered solution of the ribozymes, EGS molecules, DNA encoding ribozymes, EGS molecules or antisense to be encapsulated, and the resulting hydrated lipid vesicles or liposomes encapsulating the material can then be washed by centrifugation and can be filtered and stored at 4°C. This method has been used to deliver nucleic acid molecules to the nucleus and cytoplasm of cells of the MOLT-3 leukemia cell line (Thierry, A.R. and Dritschilo, A., Nucl. Acids Res . , 20 : 5691-5698 (1992)) . Alternatively, ribozymes, EGS, antisense molecules, or DNA encoding such molecules, can be incorporated within microparticles, or bound to the outside of the microparticles, either ionically or covalently.
Cationic liposomes or microcapsules are microparticles that are particularly useful for delivering negatively charged compounds such as nucleic acid-based compounds, which can bind ionically to the positively charged outer surface of these liposomes. Various cationic liposomes have previously been shown to be very effective at delivering nucleic acids or nucleic acid-protein complexes to cells both in vi tro and in vivo, as reported by Feigner, P.L. et al . , Proc . Natl . Acad. Sci . USA, 84 : 7413-7417 (1987); Feigner, P.L., Advanced Drug Delivery Reviews, 5 : 163-187 (1990) ; Clarenc, J.P. et al., Anti -Cancer Drug Design, 8 : 81-94 (1993) , incorporated herein by reference. Cationic liposomes or microcapsules can be prepared using mixtures including one or more lipids containing a cationic side group in a sufficient quantity such that the liposomes or microcapsules formed from the mixture possess a net positive charge which will ionically bind negatively charged compounds. Examples of positively charged lipids that may be used to produce cationic liposomes include the aminolipid dioleoyl phosphatidyl ethanolamine (PE) , which possesses a positively charged primary amino head group; phosphatidylcholine (PC) , which possess positively charged head groups that are not primary amines; and N[1- (2, 3-dioleyloxy)propyl] -N,N,N- triethylammonium ( "DOTMA, " see Feigner, P.L. et al., Proc . Natl . Acad. Sci USA, 84 , 7413-7417 (1987) ; Feigner, P.L. et al. , Nature, 337, 387-388 (1989) ; Feigner, P.L., Advanced Drug Delivery Reviews, 5, 163-187 (1990)) . Nucleic acid can also be encapsulated by or coated on cationic liposomes which can be injected intravenously into a mammal . This system has been used to introduce DNA into the cells of multiple tissues of adult mice, including endothelium and bone marrow, where hematopoietic cells reside (see, for example, Zhu et al . , Science, 261 : 209-211 (1993) ) .
Liposomes containing either ribozymes, EGS, antisense molecules or DNA encoding these molecules, can be administered systemically, for example, by intravenous or intraperitoneal administration, in an amount effective for delivery of the anti-cancer-associated mRNA ribozyme, EGS or antisense to targeted cells. Other possible routes include trans-dermal or oral, when used in conjunction with appropriate microparticles. Generally, the total amount of the liposome- associated nucleic acid administered to an individual will be less than the amount of the unassociated nucleic acid that must be administered for the same desired or intended effect.
Compositions including various polymers such as the polylactic acid and polyglycolic acid copolymers, polyethylene, and polyorthoesters and the anti-APL ribozymes, EGS, antisense molecules, or DNA encoding such molecules, can be delivered locally to the appropriate cells by using a catheter or syringe. Other means of delivering such compositions locally to cells include using infusion pumps (for example, from Alza Corporation, Palo Alto, California) or incorporating the compositions into polymeric implants (see, for example, P. Johnson and J.G. Lloyd-Jones, eds., Drug Delivery Systems (Chichester, England: Ellis Horwood Ltd., 1987) , which can effect a sustained 35 release of the therapeutic anti-APL ribozyme or EGS compositions to the immediate area of the implant.
The following examples are presented for illustrative purposes and additional guidance. EXAMPLES
Example 1: Plasmids, Oligonucleotide Synthesis and Transcription Reactions for Construction and Analysis of Anti- APL mRNA Molecules. Plasmids: Plasmid pAPL 7-5, diagrammed in
Figure 8, was constructed by cloning a 788 nucleotide fragment spanning the PML-RARα fusion region (nt 1060 to 1848 of SEQ ID NO. 1, corresponding to a PML sequence of nucleotides 1076-1739 of clone B16 and a RARα sequence of nucleotides 1766-1890 of PML-RARα clone B467 of de The, et al . , Cell , 66 : 675-684 (1991)) in the vector pCRlOOO (Invitrogen Corp., San Diego, CA) . This fragment was PCR amplified from total mRNA of a cell line whose breakpoint and sequence are identical to that of the NB4 cell line (de The, et al . , Cell , 66 : 675-684 (1991) , Lanotte, M. et al . , Blood, 77 : 1080-1086 (1991)) . The sequence in the fusion region was verified to be identical to that previously reported (de The, et al . , Cell , 66 : 675- 684 (1991) ) . An EcoRI/Hindlll restriction fragment from this plasmid was cloned into the vector pGEM™- 3Z (Promega, Madison, Wisconsin) to generate plasmid pAPL-3Z3. Plasmid pRAR5 was constructed by cloning an J57coRI fragment of a plasmid containing the full length RARα sequence from an M13 sequencing vector into the EcoRI cloning site of pGEM™-3Z (Promega, Madison, Wisconsin) . The desired construct had the RARα coding sequence (see SEQ ID NO. 2) cloned downstream of the T7 promoter and was selected by restriction analysis. For transcription, this plasmid was linearized with AccI .
Oligonucleotides: All the oligonucleotides used for transcription and sequencing were synthesized on an Applied Biosystems, Inc. (ABI) DNA synthesizer model 392. Gel-purified oligodeoxyribonucleotides were resuspended in 10 mM Tris HCI, pH 8.0, 1 mM EDTA and stored at -20°C. Ribozymes and EGS molecules were synthesized in vi tro by transcription with T7 RNA polymerase . Transcriptions; Run-off transcriptions of linearized plasmids (2.5 μg) were performed in 100 μl reactions containing 40 mM Tris HCI, pH 7.5, 18 mM MgCl2, 1 mM spermidine, 5 mM DTT, 2000 U/ml placental RNase inhibitor (Promega) , 3 mM each ATP, UTP, CTP and GTP, 50 μCi of α- [32P] -rNTP (usually CTP, New England Nuclear) and 3000 U/ml of T7 RNA polymerase (New England Biolabs) . Transcription of Hindlll-linearized pAPL-3Z3 generated a transcript containing 788 nucleotides of PML-RARα and approximately 60 nucleotides of vector sequences at the 3' end while transcription of Accl-linearized pRAR5 generated a 960 nucleotide transcript. Transcription from oligonucleotides was carried out using a standard method essentially as described by Milligan, et al . {Nucl . Acids Res . , 15 : 8783-8798 (1987)) , using a complete coding strand and a partial complementary strand spanning the promoter region. All the transcriptions were carried out for 2-16 hrs at 37°C and terminated by the addition of 120 μl of a termination cocktail (formamide, EDTA and tracing dye) . The reaction mixes were then heated at 90°C for 3 minutes, snap-cooled in ice, and subjected to gel electrophoresis on urea/polyacryla ide gels. Plasmid pAPL-3Z3 expressing PML-RARα Jbcrl DNA generated a 788 nt (nt 1060 to 1848 of Sequence ID NO. 3) RNA transcript containing 664 nt of the PML sequence (up to the fusion junction, nt 1060 to 1724 of Sequence ID NO. 3) and 124 nucleotides of the RARα sequence (starting with the fusion junction, nt 1724 to 1848 of Sequence ID NO. 3) . When linearized with the restriction enzyme AccI, the plasmid expressing the RARα sequence generates a 960 nt transcript, corresponding to nt 1 to 960 of SEQ ID NO. 4. The site where the PML-RARα fusion occurs in the recombined gene is located at nt 146 of this sequence.
The transcription products were visualized by ultraviolet light shadowing and the appropriate bands excised and eluted from the polyacrylamide gels. The purified RNAs were resuspended in water and stored at -20°C. Ribozyme Cleavage Assay; Cleavage reactions were carried out in a volume of 10 μl in 50 mM Tris HCI, pH 7.5, 30 mM MgCl2, for 3 hours at 37°C. The reactions contained 0.03 μM of the radiolabeled RNA substrates and varying amounts of ribozyme (0,0.03, 0.1, 0.3, 1, 3 and 6 μM) . The reactions were stopped by adding 10 μl of a stop solution (formamide containing 30 mM EDTA and tracing dyes 0.025% bromophenol blue and 0.025% xylene cyanol) , followed by heating at 90°C'for 3 minutes. The reaction products were separated on 4% polyacrylamide gels containing 7 M urea.
The bands corresponding to the precursor RNA substrate and the resulting two cleavage products were counted from the dried gel using a Betascope gel analyzer (Betagen) . The percentage of precursor RNA substrate remaining was plotted as function of the ribozyme concentration and the catalytic efficiency expressed as k^,./!^ (where kcat is the rate constant of cleavage and K,,, is the Michaelis constant) , the second order rate constant for the reaction of free ribozyme and substrate. Following the methods of Heidenreich and Eckstein (J. Biol . Chem. , 267 : 1904-1909 (1992) , the efficiency of the cleavage reaction, k^/I,.) , was determined using the formula
-In F/t=(kcat/K [C] where F is the fraction of RNA substrate left, t is the reaction time, and [C] is the ribozyme concentration.
Example 2: Construction and Testing of
Alternative Anti-APL-Specific mRNA Ribozymes.
Anti-APL ribozymes were synthesized by in vitro transcription using T7 RNA polymerase. Ribozymes were designed and synthesized to preferentially bind APL-associated PML-RARα mRNA sequences associated with APL. There are two hammerhead ribozyme cleavage sites in the vicinity of the bcrl fusion junction: cleavage site 1 is an AUU located two nucleotides 3' to the PML-RARα fusion sequence (nt 1727 to 1729 of Sequence ID No. 3) and cleavage site 2 is a UUC located twenty-two nucleotides 3' to the fusion (nt 1747 to 1749 of SEQ ID NO. 3) . Both of these cleavage sites are actually located in the RARα portion of the PML- RARα sequence .
Ribozyme Construct IHRZl.18: Ribozyme construct IHRZl.18 (SEQ ID NO. 5) was designed to target cleavage site 1 of the PML-RARα fusion sequence. As shown in Figure la, the guide sequence (nt 29 to 36 SEQ ID NO. 5) of one of the arms of IHRZl.18 is complementary to an eight nucleotide sequence at and across the junction of the PML-RARα fusion (nt 1721 to 1728 of SEQ ID No. 3) and forms helix III upon binding to the PML-RARα substrate RNA molecule (SEQ ID NO. 3) (see Figure la) . Only four base pairs can form in helix III when IHRZl.18 is bound to the wild-type RARα substrate RNA (nt 142 to 150 of SEQ ID NO. 4) (see Figure lb) . The guide sequence (nt 1 to 6 of SEQ ID NO. 5) of the other arm of IHRZl.18 is complementary to the RARα sequence of six nucleotides immediately 3' of cleavage site 1 of the PML-RARα RNA (nt 1730 to 1735 of Sequence ID No. 3) and forms helix I upon binding to either the PML-RARα or the wild-type RARα substrate RNA molecules (Figures la and lb) . Thus, binding of IHRZl.18 to the RARα RNA sequence, instead of PML- RARα RNA, results in the loss of at least two base pairs, with a further destabilization caused by the presence of bulging unpaired nucleotides in the middle of the helix III region (see Figure lb) . Thus, it is predicted by plain energetic considerations that IHRZl.18 will have a preference for the PML-RARα substrate RNA over the RARα RNA. Ribozyme Construct IHRZl.3: Ribozyme construct IHRZl.3 (SEQ ID NO. 6) was designed to target cleavage site 2 of the PML-RARα fusion sequence. A portion of the guide sequence (nt 34 to 41 of SEQ ID NO. 6) of one arm of IHRZl .3 is complementary to and binds to an eight nucleotide sequence (nt 1721 to 1728 of SEQ ID NO. 3) at and across the junction of the PML-RARα fusion RNA sequence (Figure 2a) . The same guide sequence is also complementary to a three nucleotide sequence 5' of cleavage site 2 and present in both the PML- RARα and the RARα substrate RNA molecules (Figures 2a and 2b) . Binding of this guide sequence to PML- RARα substrate RNA results in a helix III interrupted by a looping out of seventeen nucleotides (nt 1729 to 1745 of SEQ ID NO. 3) of the PML-RARα substrate RNA opposite a three nucleotide loop out of the IHRZl.3 construct (Figure 2a) . As shown in Figure 2a, the portion of the interrupted helix III 5' of the seventeen nucleotide loop out is designated helix Illb and consists of the eight base pairs found in helix III of IHRZl.18 (Figure la) . The portion of helix III 3' from the seventeen nucleotide loop out of PML- RARα substrate RNA is designated helix Ilia and consists of the above-mentioned three base pairs 5' of cleavage site 2. Binding of this guide sequence in IHRZl.3 to wild-type RARα substrate RNA also results in an interrupted helix III (helices Ilia and Illb) in which helix Illb is formed by only four, instead of eight, base pairs 5' of the seventeen nucleotide loop out of the RARα substrate RNA (Figure 2b) .
Helix 1 of the ribozyme-substrate complex is formed between another arm of IHRZl.3 consisting of a five nucleotide guide sequence (nt 1 to nt 5 of SEQ ID NO. 6) and a sequence of the substrate RNA
(nt 1750 to nt 1754 of SEQ ID NO. 3) (Figures 2a and 2b) .
Ribozyme Construct IHRZl.30: Ribozyme construct IHRZl.30 (SEQ ID NO. 7) is designed to target cleavage site 1 of the PML-RARα fusion sequence. One portion of the guide sequence (nt 34 to 41 of SEQ ID NO. 7) of one arm of IHRZl.30 is complementary to eight nucleotides 5' of the PML- RARα fusion junction (nt 1701 to 1708 of Sequence ID No. 3) and another portion of the same guide sequence is complementary to three nucleotides 5' of cleavage site 2 (3' to the fusion junction) (see Figure 3a) . Binding of this guide sequence to the PML-RARα substrate RNA (nt 1701 to 1728 SEQ ID NO. 3) , as shown in Figure 3a, forms a helix III interrupted by a looping out of 17 nucleotides (nt 1709 to 1725 of Sequence ID No. 3) of the PML-RARα substrate RNA opposite a three nucleotide loop out of the IHRZl.30 sequence. Helix Illb is formed by the complementary base pairing between the eight bases of the guide sequence and of the PML-RARα sequence 5' of the 17 nucleotide loop out, and helix Ilia is formed by the base pairing between three other nucleotides of the guide sequence and those of the PML-RARα sequence 3' of the 17 nucleotide loop out (see Figure 3a) .
The complex that IHRZl.30 can form with RARα is different from the one formed by PML-RARα, as shown in Figure 3b. This complex results in the formation of a stem between nt 35 to 38 of SEQ ID NO. 7 and nt 139-142 of SEQ ID NO. 2 (helix Illb) and a stem between nt 29 to 31 of SEQ ID NO. 7 and nt 147 to 149 of SEQ ID NO. 4 (helix Ilia) . Helix
I remains as in the PML-RARα complex.
Example 3: Screening for Anti-APL-Associated RNA Ribozyme Activity.
The ribozyme constructs described in Example 2 were assayed using the standard cleavage assay described in Example 1 to determine the efficiency of the cleavage reaction against a PML-RARα substrate RNA and against an RARα substrate RNA.
The data is presented Figures 4 to 6 and in Table I below.
The efficiency (kcat/K of cleavage of the PML- RARα and the RARα RNA substrate molecules for each of the hammerhead ribozyme constructs is given in Table I. Table I: Catalytic Efficiencies of Ribozymes
Ribozyme Substrate PML-RARα RARα kcat/Km Ratio (m"1 S"1)
IHRZl.18 560.2 82.1 6.8
IHRZl.3 164.0 2.3 71.3
IHRZl.30 19.5 50.7 0.38
Construct IHRZl.18 efficiently cleaved both the PML-RARα RNA and the RARα substrate RNA molecules (Table I) . At lower concentrations, IHRZl.18 is quite selective for the PML-RARα fusion RNA, but this selectivity tapers off at higher concentrations of the ribozyme (Figure 4) .
In contrast, ribozyme IHRZl.3 displayed approximately four-fold lower activity towards site 2 of the PML-RARα fusion RNA substrate than did IHRZl.18 (a kcat/K,„ of 164 M"1 s"1 for IHRZl .3 compared with 560 M"1 s'1 for IHRZl.18 in Table I. However, compared to IHRZl.18, IHRZl.3 appeared to be much more specific for the PML-RARα RNA than the RARα transcript for which it displayed a kcat/K,_ of only 2.3 M"1 s"1 (see Table I) . Compared to the other constructs, cleavage products from IHRZl.3 activity against RARα RNA were barely detectable on gels. Even at the highest concentration of ribozyme used (6 μM) , the extent of cleavage of the RARα RNA was less than 20% (see Figure 5) . Thus, although IHRZl.3 exhibited a four-fold lower activity against PML-RARα than IHRZl.18, it typically was forty to eighty-fold more active with PML-RARα substrate RNA than with RARα substrate RNA as indicated by the ratio of its efficiency with PML-RARα and RARα substrate RNA molecules (see "Ratio" column, Table I) .
Ribozyme construct IHRZl.30, a modified ribozyme modeled on the design of IHRZl .3, except directed to site 1 on the PML-RARα RNA, actually cleaved the wild-type RARα substrate RNA two to three-fold better than the PML-RARα substrate RNA (Figure 6 and Table I) . Construct IHRZl.30 turned out to be a very weak ribozyme, and, as expected, it actually cleaved the RARα mRNA better than the PML-RARα mRNA (Figure 6 and Table I) .
Measurement of the efficiency (kcat/K for the reaction of a ribozyme construct with the cancer- associated PML-RARα RNA sequence and the wild-type RARα RNA sequence clearly allows a more accurate mathematical description of the catalytic behavior of the ribozymes. As shown in Table I, IHRZl.18 is 6.8 times more active against the cancer-associated PML-RARα fusion mRNA sequence than against the wild-type RARα mRNA sequence. However, IHRZl .3 is 71.3 times more active against the cancer- associated fusion mRNA sequence compared to the RARα mRNA sequence. As expected, the ratio of the efficiency of cleavage of PML-RARα substrate RNA sequence and of RARα substrate RNA sequence by the IHRZl.30 ribozyme was extremely low (0.38, Table I) , reflecting this ribozyme's greater efficiency at cleaving the wild-type RARα substrate RNA than the PML-RARα substrate RNA. IHRZl.18 and IHRZl .3 complement each other in their selectivity behavior, in different concentration ranges (Figures 4 and 5) . With regard to their therapeutic use, it would be preferable to use the most active ribozyme, at concentrations where the best selectivity can be achieved. However, as most ribozyme-dependent inactivation studies have shown, high doses are often required to inactivate mRNA molecules, and ribozyme synthesis must be driven by powerful promoters, as described above. Thus, a ribozyme such as IHRZl.3, which displays selectivity at high concentrations, may be a better choice.
Example 4: Synthesis of anti-APL mRNA
Phosphorothioate Antisense Molecule.
Anti-APL phosphorothioate antisense molecules complementary to the fusion junction of the PML- RARα mRNA were synthesized on an automated oligonucleotide synthesizer by QCB Inc., Hopkinton, MA. These oligonucleotides were deprotected and desalted on a G25 gel filtration column. Four oligonucleotides sequences were synthesized.
Oligonucleotide As-APL 1 hybridizes to nucleotides 1713-1737 of PML-RARα SEQ ID 1; As-APL2 hybridizes to nucleotides 1710-1734 of SEQ ID 1 and As-APL3 hybridizes to nucleotides 1716-1740 of SEQ ID 1. A control sense oligonucleotide, AS-APL4, which has the same sequence as the fusion junction (nucleotides 1716-1714 of SEQ ID 1) was also synthesized. All antisense molecules are capable of binding to the fusion junction of the PML-RARα mRNA. These molecules when introduced into cells expressing the PML-RARα mRNA should inhibit the production of the PML-RARα hybrid protein and can thus relieve the maturation block of the APL cells and cause them to differentiate from the promyelocyte to the granulocyte. These phosphorothioate molecules can either be delivered directly to APL cells in culture or can be complexed with cationic lipids and then delivered to the cells. Sequences of anti-APL phosphorothioate antisense: AS-APL1: 5' -TGGGTCTCAATGGCTGCCTCCCCGG AS-APL2 : 5' -GTCTCAATGGCTGCCTCCCCGGCGC AS-APL3 : 5' -CTCTGGGTCTCAATGGCTGCCTCCC AS-APL4 : 5' -CCGGGGAGGCAGCCATTGAGACCCA Example 5: Proof of efficacy of hammerhead ribozyme in APL cells. cDNAs encoding a series of hammerhead ribozymes APL 1.0,1.1, 5.0 (Figure 9) (SEQ ID NO. 12) based on IDRZ 1.18 (Figure la) were synthesized and were cloned into Eboplpp vector, an Epstein- Barr-based episomally replicating vector under the control of an SV40 promoter and encoding a hygromycin selectable gene (Mossar M.M. et al, Oncogene, 9, 833-840 (1994)) sandwiched between two self cleaving hammerhead ribozymes as described by Tiara et al . , (Nucleic Acid Res . 19, 5125 (1991)) . Upon transcription of the construct, the self- cleaving ribozymes are expected to release the APL- specific ribozymes. APL 1.0 had 16 nucleotide hybridizing arms while APL 1.1 had 29 nucleotide hybridizing arms and both ribozymes were designed to cleave the same site. APL 5.0 was analogous to APL 1.0 except that it had a two nucleotide deletion in the catalytic core of the ribozyme thereby making it catalytically inactive (Figure 9) . Another series of cDNAs encoding ribozymes based on IDRZ 1.3 (Figure 2a) named APL 2.0, 2.1, 6.0 and 6.1 (Figure 10) were also synthesized and cloned into Eboplpp vector. APL 2.0 had a 16 nucleotide hybridizing arms while APL 2.1 had 30 nucleotide hybridizing arms. APL 6.0 and 6.1 were analogous to 2.0 and 2.1 except that it had a two nucleotide deletion in the catalytic core of the ribozyme thereby making it catalytically inactive (Figure 10) . In order to test whether the self-cleaving hammerhead ribozymes flanking the APL ribozymes did indeed self-cleave and release the APL ribozymes between them, all APL ribozymes constructs with the flanking self-cleaving ribozymes, were cloned into a pGEM vector (Promega Corp. Madison, Wl, USA) under the control of a T7 RNA polymerase promoter and transcribed in a test tube using T7 RNA polymerase in presence of 32P ATP according to the instructions of the manufacturer. The reaction products were run on a polyacrylamide gel and the radioactive bands were visualized using a phosphoimager. As expected, the self-cleaving hammerhead ribozymes released APL ribozymes from the construct.
The ribozymes bearing Eboplpp vectors, APL 1.0, APL 1.1, APL 2.0, APL 2.1 (SEQ ID NO. 14) , and the controls APL 5 and APL 6.1 were transfected into NB4 cells, a human APL cell line (M. Lanotte, et. al., Blood 77, 1080 (1991)) using electroporation techniques (Mossar M.M. , et. al., Oncogene, 9, 833 (1994)) . The transfected cells were selected under low hygromycin (65 μg ml) . Upon increasing the level of hygromycin, the episomal copy number is expected to increase (Mossar M.M., et . al . , Oncogene, 9, 833 (1994)) thereby increasing the expression of the various ribozymes in cells. After hygromycin-resistant cells were selected, the hygromycin dose was increased to 500 μg/ml culture media in order to increase the expression of the ribozymes. After seven days the proliferative potential of the cells were assayed by the MTT proliferation assay (Mosmann T et . al . , Journal of Immunological Methods , 65, 55 (1983)) . The active ribozyme- bearing constructs APL 2.0 and 2.1 were severely inhibitory to cell growth whereas APL 5, which had the same hybridizing arms but had no catalytic activity had no effect on cell growth (Figure 11) demonstrating that the inhibition was not the result of an antisense block of the translation of the APL RNA, but rather through specific cleavage of the fusion junction. This result also indicated that the growth inhibitory activity at higher levels of hygromycin was not caused by the drug. None of the constructs had any effect on cell growth at 65 μg/ml of hygromycin. The results indicate that the expression of ribozymes capable of cleaving the PML-RAP junction is severely inhibitory to cell growth. APL 2.0 which has a shorter hybridizing arm compared with APL.2.1 was less inhibitory to cell growth as predicted. It was further observed that prolonged expression
(greater than 7 days) of the more potent APL 2.1 ribozyme at higher levels (500 μg hygromycin) resulted in cell death as measured and it was observed that both 1.0 and 1.1 which showed higher cleavage activity in test tube was more potent inhibitors of NB4 cell growth with inhibition observed three days after increasing the hygromycin.
In order to further characterize the inhibitory activity, increasing levels of hygromycin was added to cells expressing APL 2.1 ribozyme. As seen in Figure 12, a clear dose response was observed with increasing hygromycin concentration. A 130 μg dose was only partially effective in inhibiting cell growth while higher concentrations inhibited cell growth in a dose- dependent manner. The slight inhibition of cell growth on day 13 observed at 65 μg concentration of hygromycin was due to overcrowding of cells resulting in their partial death. Expression of the ribozymes in BEAS2 cells, a teratocarcinoma cell line, had no effect on their growth, indicating that the anti-proliferative effect of the active ribozymes on NB4 cells may be directly through the inactivation of APL RNA. Example 6 : Proof of efficacy of APL EGSs Synthesis of EGSs:
Two EGSs APL A 20 (SEQ ID NO. 15) and APL 1009 (SEQ ID NO. 16) targeted to the fusion junction of PML RAR were chemically synthesized on an Applied Biosystems 394 DNA/RNA synthesizer. The sequence of these EGSs and their chemical composition are shown in Figures 13a and 13c. EGS A20D which lacked two nucleotide in the sequences corresponding to the T-loop of the EGS but was otherwise similar to A20 is shown in Figure 13b. EGS APL 1017, shown in Figure 13d, lacked three nucleotides in the T-loop but was otherwise similar to APL 1009. The control EGSs (A20D and APL 1017) were incapable of inducing cleavage of APL mRNA in presence of RNaseP and but could hybridize to the fusion junction. The EGSs were purified by Reverse-phase HPLC, concentrated, and suspended in 2M NaCl to convert the EGS into the Na form and dialyzed extensively against water and then lyophilized. The EGSs were suspended in water for test tube cleavage assay or in 150 mM NaCl for cell culture testing.
Test tube cleavage assay: 3 ng of linearized pAPL 7-5 plasmid with HindlLI restriction enzyme was transcribed as described in Example 1 in presence of 32P-ATP for 30 min. 0.25 μM (final concentration) of EGS and 2 μl of a purified preparation of RNase P from HeLa cells (Bartkiewicz, M. et. al., Genes and
Development , 3, 488-499 (1989)) was added to the transcription reaction during the transcription. The reaction products were separated on a denaturing polyacrylamide gel and visualized using a Molecular Dynamics Phosphoimager. Both A20 and APL 1009 induced cleavage of the APL RNA at the fusion junction, while A20D and APL 1017 were incapable of inducing cleavage of APL RNA.
Cell culture testing: As shown in both EGSs A20 (Figure 14a) and APL 1009 (Figure 15a) were inhibitory to cell growth as measured by MTT assay while the corresponding inactive controls A20D (Figure 14b) and APL 1007 (Figure 15b) had no effect on cell growth. Both A20 and APL 1009 showed dose dependent inhibition of NB4 cell growth with observed above 3 μM concentration.
Modifications and variations of the method of the present invention will be obvious to those skilled in the art from the foregoing detailed description. Such modifications and variations are intended to come within the scope of the appended claims .
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Innovir Laboratories, Inc. (ii) TITLE OF INVENTION: Ribozyme-Mediated Inactivation of Leukemia-Associated RNA
(iii) NUMBER OF SEQUENCES: 16 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Patrea L. Pabst
(B) STREET: 2800 One Atlantic Center
1201 West Peachtree Street
(C) CITY: Atlanta
(D) STATE: Georgia
(E) COUNTRY: USA
(F) ZIP: 30309-3450 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/354,956
(B) FILING DATE: 14-DEC-1994
(C) CLASSIFICATION: (viii) ATTORNE /AGENT INFORMATION:
(A) NAME: Pabst, Patrea L. <B) REGISTRATION NUMBER: 31,284 (C) REFERENCE/DOCKET NUMBER: ILI110PCT (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (404) 873-8794
(B) TELEFAX: (404) 873-8795
(2) INFORMATION FOR SEQ ID NO:1 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3511 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOG : linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..3511
(D) OTHER INFORMATION: /function= "PML-RARα DNA Sequence." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
CTCCCCTTCA GCTTCTCTTC ACGCACTCCA AGATCTAAAC CGAGAATCGA AACTAAGCTG 60
GGGTCCATGG AGCCTGCACC CGCCCGATCT CCGAGGCCCC AGCAGGACCC CGCCCGGCCC 120
CAGGAGCCCA CCATGCCTCC CCCCGAGACC CCCTCTGAAG GCCGCCAGCC CAGCCCCAGC 180
CCCAGCCCTA CAGAGCGAGC CCCCGCTTCG GAGGAGGAGT TCCAGTTTCT GCGCTGCCAG 240
CAATGCCAGG CGGAAGCCAA GTGCCCGAAG CTGCTGCCTT GTCTGCACAC GCTGTGCTCA 300
GGATGCCTGG AGGCGTCGGG CATGCAGTGC CCCATCTGCC AGGCGCCCTG GCCCCTAGGT 360
GCAGACACAC CCGCCCTGGA TAACGTCTTT TTCGAGAGTC TGCAGCGGCG CCTGTCGGTG 420 TACCGGCAGA TTGTGGATGC GCAGGCTGTG TGCACCCGCT GCAAAGAGTC GGCCGACTTC 480
TGGTGCTTTG AGTGCGAGCA GCTCCTCTGC GCCAAGTGCT TCGAGGCACA CCAGTGGTTC 540
CTCAAGCACG AGGCCCGGCC CCTAGCAGAG CTGCGCAACC AGTCGGTGCG TGAGTTCCTG 600
GACGGCACCC GCAAGACCAA CAACATCTTC TGCTCCAACC CCAACCACCG CACCCCTACG 660
CTGACCAGCA TCTACTGCCG AGGATGTTCC AAGCCGCTGT GCTGCTCGTG CGCGCTCCTT 720
GACAGCAGCC ACAGTGAGCT CAAGTGCGAC ATCAGCGCAG AGATCCAGCA GCGACAGGAG 780
GAGCTGGACG CCATGACGCA GGCGCTGCAG GAGCAGGATA GTGCCTTTGG CGCGGTTCAC 840
GCGCAGATGC ACGCGGCCGT CGGCCAGCTG GGCCGCGCGC GTGCCGAGAC CGAGGAGCTG 900
ATCCGCGAGC GCGTGCGCCA GGTGGTAGCT CACGTGCGGG CTCAGGAGCG CGAGCTGCTG 960
GAGGCTGTGG ACGCGCGGTA CCAGCGCGAC TACGAGGAGA TGGCCAGTCG GCTGGGCCGC 1020
CTGGATGCTG TGCTGCAGCG CATCCGCACG GGCAGCGCGC TGGTGCAGAG GATGAAGTGC 1080
TACGCCTCGG ACCAGGAGGT GCTGGACATG CACGGTTTCC TGCGCCAGGC GCTCTGCCGC 1140
CTGCGCCAGG AGGAGCCCCA GAGCCTGCAA GCTGCCGTGC GCACCGATGG CTTCGACGAG 1200
TTCAAGGTGC GCCTGCAGGA CCTCAGCTCT TGCATCACCC AGGGGAAAGA TGCAGCTGTA 1260
TCCAAGAAAG CCAGCCCAGA GGCTGCCAGC ACTCCCAGGG ACCCTATTGA CGTTGACCTG 1320
CCCGAGGAGG CAGAGAGAGT GAAGGCCCAG GTTCAGGCCC TGGGGCTGGC TGAAGCCCAG 1380
CCTATGGCTG TGGTACAGTC AGTGCCCGGG GCACACCCCG TGCCAGTGTA CGCCTTCTCC 1440
ATCAAAGGCC CTTCCTATGG AGAGGATGTC TCCAATNACA ACGACAGCCC AGAAGAGGAA 1500
GTGCAGCCAG ACCCAGTGCC CCAGGAAGGT CATCAAGATG GAGTCTGAGG AGGGGAAGGA 1560
GGCAAGGTTG GCTCGGAGCT CCCCGGAGCA GCCCAGGCCC AGCACCTCCA AGGCAGTCTC 1620
ACCACCCCAC CTGGATGGAC CGCCTAGCCC CAGGAGCCCC GTCATAGGAA GTGAGGTCTT 1680
CCTGCCCAAC AGCAACCACG TGGCCAGTGG CGCCGGGGAG GCAGCCATTG AGACCCAGAG 1740
CAGCAGTTCT GAAGAGATAG TGCCCAGCCC TCCCTCGCCA CCCCCTCTAC CCCGCATCTA 1800
CAAGCCTTGC TTTGTCTGTC AGGACAAGTC CTCAGGCTAC CACTATGGGG TCAGCGCCTG 1860
TGAGGGCTGC AAGGGCTTCT TCCGCCGCAG CATCCAGAAG AACATGGTGT ACACGTGTCA 1920
CCGGGACAAG AACTGCATCA TCAACAAGGT GACCCGGAAC CGCTGCCAGT ACTGCCGACT 1980
GCAGAAGTGC TTTGAAGTGG GCATGTCCAA GGAGTCTGTG AGAAACGACC GAAACAAGAA 2040
GAAGAAGGAG GTGCCCAAGC CCGAGTGCTC TGAGAGCTAC ACGCTGACGC CGGAGGTGGG 2100
GGAGCTCATT GAGAAGGTGC GCAAAGCGCA CCAGGAAACC TTCCCTGCCC TCTGCCAGCT 2160
GGGCAAATAC ACTACGAACA ACAGCTCAGA ACAACGTGTC TCTCTGGACA TTGACCTCTG 2220
GGACAAGTTC AGTGAACTCT CCACCAAGTG CATCATTAAG ACTGTGGAGT TCGCCAAGCA 2280
GCTGCCCGGC TTCACCACCC TCACCATCGC CGACCAGATC ACCCTCCTCA AGGCTGCCTG 2340
CCTGGACATC CTGATCCTGC GGATCTGCAC GCGGTACACG CCCGAGCAGG ACACCATGAC 2400 52
CTTCTCGGAC GGGCTGACCC TGAACCGGAC CCAGATGCAC AACGCTGGCT TCGGCCCCCT 2460
CACCGACCTG GTCTTTGCCT TCGCCAACCA GCTGCTGCCC CTGGAGATGG ATGATGCGGA 2520
GACGGGGCTG CTCAGCGCCA TCTGCCTCAT CTGCGGAGAC CGCCAGGACC TGGAGCAGCC 2580
GGACCGGGTG GACATGCTGC AGGAGCCGCT GCTGGAGGCG CTAAAGGTCT ACGTGCGGAA 2640
GCGGAGGCCC AGCCGCCCCC ACATGTTCCC CAAGATGCTA ATGAAGATTA CTGACCTGCG 2700
AAGCATCAGC GCCAAGGGGG CTGAGCGGGT GATCACGCTG AAGATGGAGA TCCCGGGCTC 2760
CATGCCGCCT CTCATCCAGG AAATGTTGGA GAACTCAGAG GGCCTGGACA CTCTGAGCGG 2820
ACAGCCGGGG GGTGGGGGGC GGGACGGGGG TGGCCTGGCC CCCCCGCCAG GCAGCTGTAG 2880
CCCCAGCCTC AGCCCCAGCT CCAACAGAAG CAGCCCGGCC ACCCACTCCC CGTGACCGCC 2940
CACGCCACAT GGACACAGCC CTCGCCCTCC GCCCCGGCTT TTCTCTGCCT TTCTACCGAC 3000
CATGTGACCC CGCACCAGCC CTGCCCCCAC CTGCCCTCCC GGGCAGTACT GGGGACCTTC 3060
CCTGGGGGAC GGGGAGGGAG GAGGCAGCGA CTCCTTGGAC AGAGGCCTGG GCCCTCAGTG 3120
GACTGCCTGC TCCCACAGCC TGGGCTGACG TCAGAGGCCG AGGCCAGGAA CTGAGTGAGG 3180
CCCCTGGTCC TGGGTCTCAG GATGGGTCCT GGGGGCCTCG TGTTCATCAA GACACCCCTC 3240
TGCCCAGCTC ACCACATCTT CATCACCAGC AAACGCCAGG ACTTGGCTCC CCCATCCTCA 3300
GAACTCACAA GCCATTGCTC CCCAGCTGGG GAACCTCAAC CTCCCCCCTG CCTCGGTTGG 3360
TGACAGAGGG GGTGGGACAG GGGCGGGGGG TTCCCCCTGT ACATACCCTG CCATACCAAC 3420
CCCAGGTATT AATTCTCGCT GGTTTTGTTT TTATTTTAAT TTTTTTGTTT TGATTTTTTT 3480
AATAAGAATT TTCATTTTAA GCAAAAAAAA A 3511
(2) INFORMATION FOR SEQ ID NO:2 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1481 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..1481
(D) OTHER INFORMATION: /function= "RARα DNA Sequence."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2 :
GGGGGCGGGC ACCTCAATGG GTACCCGGTG CCTCCCTACG CCTTCTTCTT CCCCCCTATG 60
CTGGGTGGAC TCTCCCCGCC AGGCGCTCTG ACCACTCTCC AGCACCAGCT TCCAGTTAGT 120
GGATATAGCA CACCATCCCC AGCCACCATT GAGACCCAGA GCAGCAGTTC TGAAGAGATA 180
GTGCCCAGCC CTCCCTCGCC ACCCCCTCTA CCCCGCATCT ACAAGCCTTG CTTTGTCTGT 240
CAGGACAAGT CCTCAGGCTA CCACTATGGG GTCAGCGCCT GTGAGGGCTG CAAGGGCTTC 300
TTCCGCCGCA GCATCCAGAA GAACATGGTG TACACGTGTC ACCGGGACAA GAACTGCATC 360 ATCAACAAGG TGACCCGGAA CCGCTGCCAG TACTGCCGAC TGCAGAAGTG CTTTGAAGTG 420
GGCATGTCCA AGGAGTCTGT GAGAAACGAC CGAAACAAGA AGAAGAAGGA GGTGCCCAAG 480
CCCGAGTGCT CTGAGAGCTA CACCGTGACG CCGGAGGTGG GGGAGCTCAT TGAGAAGGTG 540
CGTAAAGCGC ACCAGGAAAC CTTCCCTGCC CTCTGCCAGC TGGGCAAATC AATCACGAAC 600
AACAGCTCAG AACAACGTGT CTCTCTGGAC ATTGACCTCT GGGACAAGTT CAGTGAACTC 660
TCCACCAAGT GCATCATTAA GACTGTGGAG TTCGCCAAGC AGCTGCCCGG CTTCACCACC 720
CTCACCATCG CCGACCAGAT CACCCTCCTC AAGGCTGCCT GCCTGGACAT CCTGATCCTG 780
CGGATCTGCA CGCGGTACAC GCCCGAGCAG GACACCATGA CCTTCTCGGA CGGGCTGACC 840
CTGAACCGGA CCCAGATGCA CAACGCTGGC TTCGGCCCCC TCACCGACCT GGTCTTTGCC 900
TTCGCCAACC AGGACCGGGT GGACATGCTG CAGGAGCCGC TGCTGGAGGC GCTAAAGGTC 960
TACGTGCGGA AGCGGAGGCC CAGCCGCCCC CACATGTTCC CCAAGATGCT AATGAAGATT 1020
ACTGACCTGC GAAGCATCAG CGCCAACGGG GCTGAGCGGG TGATCACGCT GAAGATGGAG 1080
ATCCCGGGCT CCAGTCCGCC TCTCATCCAG GAAATGTTGG AGAACTCAGA GGGCCTGGAC 1140
ACTCTGAGCG GACAGCCGGG GGGTGGGGGG CGGGACGGGG GTCGCCACAT GGACACAGCC 1200
CTGGCCCTCC GCCCCCGGCT TTTCTCTGCC TTTCTACCGA CCATGTGACC CCGCACCAGC 1260
CCTGCCCCCA CCTGCCCTGC CCGGGAGTAC TGGCGACCTT CCCTGGGGGA CGGGGAGGGA 1320
GGAGGCAGCG ACTCCTTGGA CAGAGGCCTG GGCCCTCAGT GGACTGCCTG CTCCCACAGC 1380
CTGGGCTGAC GTCAGAGGCC GAGGCCAGGA ACTGAGTGAG GCCCCTGGTC CTGGGTCTCA 1440
GGATGGGTCC TGGGGGCCTC GTGTTCATCA AGACGGAATT C 1481
(2) INFORMATION FOR SEQ ID NO:3 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3511 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: RNA
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..1481
(D) OTHER INFORMATION: /function= "PML-RARα RNA Sequence."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3 :
CUCCCCUUCA GCUUCUCUUC ACGCACUCCA AGAUCUAAAC CGAGAAUCGA AACUAAGCUG 60
GGGUCCAUGG AGCCUGCACC CGCCCGAUCU CCGAGGCCCC AGCAGGACCC CGCCCGGCCC 120
CAGGAGCCCA CCAUGCCUCC CCCCGAGACC CCCUCUGAAG GCCGCCAGCC CAGCCCCAGC 180
CCCAGCCCUA CAGAGCGAGC CCCCGCUUCG GAGGAGGAGU UCCAGUUUCU GCGCUGCCAG 240
CAAUGCCAGG CGGAAGCCAA GUGCCCGAAG CUGCUGCCUU GUCUGCACAC GCUGUGCUCA 300
GGAUGCCUGG AGGCGUCGGG CAUGCAGUGC CCCAUCUGCC AGGCGCCCUG GCCCCUAGGU 360 GCAGACACAC CCGCCCUGGA UAACGUCUUU UUCGAGAGUC UGCAGCGGCG CCUGUCGGUG 420
UACCGGCAGA UUGUGGAUGC GCAGGCUGUG UGCACCCGCU GCAAAGAGUC GGCCGACUUC 480
UGGUGCUUUG AGUGCGAGCA GCUCCUCUGC GCCAAGUGCU UCGAGGCACA CCAGUGGUUC 540
CUCAAGCACG AGGCCCGGCC CCUAGCAGAG CUGCGCAACC AGUCGGUGCG UGAGUUCCUG 600
GACGGCACCC GCAAGACCAA CAACAUCUUC UGCUCCAACC CCAACCACCG CACCCCUACG 660
CUGACCAGCA UCUACUGCCG AGGAUGUUCC AAGCCGCUGU GCUGCUCGUG CGCGCUCCUU 720
GACAGCAGCC ACAGUGAGCU CAAGUGCGAC AUCAGCGCAG AGAUCCAGCA GCGACAGGAG 780
GAGCUGGACG CCAUGACGCA GGCGCUGCAG GAGCAGGAUA GUGCCUUUGG CGCGGUUCAC 840
GCGCAGAUGC ACGCGGCCGU CGGCCAGCUG GGCCGCGCGC GUGCCGAGAC CGAGGAGCUG 900
AUCCGCGAGC GCGUGCGCCA GGUGGUAGCU CACGUGCGGG CUCAGGAGCG CGAGCUGCUG 960
GAGGCUGUGG ACGCGCGGUA CCAGCGCGAC UACGAGGAGA UGGCCAGUCG GCUGGGCCGC 1020
CUGGAUGCUG UGCUGCAGCG CAUCCGCACG GGCAGCGCGC UGGUGCAGAG GAUGAAGUGC 1080
UACGCCUCGG ACCAGGAGGU GCUGGACAUG CACGGUUUCC UGCGCCAGGC GCUCUGCCGC 1140
CUGCGCCAGG AGGAGCCCCA GAGCCUGCAA GCUGCCGUGC GCACCGAUGG CUUCGACGAG 1200
UUCAAGGUGC GCCUGCAGGA CCUCAGCUCU UGCAUCACCC AGGGGAAAGA UGCAGCUGUA 1260
UCCAAGAAAG CCAGCCCAGA GGCUGCCAGC ACUCCCAGGG ACCCUAUUGA CGUUGACCUG 1320
CCCGAGGAGG CAGAGAGAGU GAAGGCCCAG GUUCAGGCCC UGGGGCUGGC UGAAGCCCAG 1380
CCUAUGGCUG UGGUACAGUC AGUGCCCGGG GCACACCCCG UGCCAGUGUA CGCCUUCUCC 1440
AUCAAAGGCC CUUCCUAUGG AGAGGAUGUC UCCAAUNACA ACGACAGCCC AGAAGAGGAA 1500
GUGCAGCCAG ACCCAGUGCC CCAGGAAGGU CAUCAAGAUG GAGUCUGAGG AGGGGAAGGA 1560
GGCAAGGUUG GCUCGGAGCU CCCCGGAGCA GCCCAGGCCC AGCACCUCCA AGGCAGUCUC 1620
ACCACCCCAC CUGGAUGGAC CGCCUAGCCC CAGGAGCCCC GUCAUAGGAA GUGAGGUCUU 1680
CCUGCCCAAC AGCAACCACG UGGCCAGUGG CGCCGGGGAG GCAGCCAUUG AGACCCAGAG 1740
CAGCAGUUCU GAAGAGAUAG UGCCCAGCCC UCCCUCGCCA CCCCCUCUAC CCCGCAUCUA 1800
CAAGCCUUGC UUUGUCUGUC AGGACAAGUC CUCAGGCUAC CACUAUGGGG UCAGCGCCUG 1860
UGAGGGCUGC AAGGGCUUCU UCCGCCGCAG CAUCCAGAAG AACAUGGUGU ACACGUGUCA 1920
CCGGGACAAG AACUGCAUCA UCAACAAGGU GACCCGGAAC CGCUGCCAGU ACUGCCGACU 1980
GCAGAAGUGC UUUGAAGUGG GCAUGUCCAA GGAGUCUGUG AGAAACGACC GAAACAAGAA 20 0
GAAGAAGGAG GUGCCCAAGC CCGAGUGCUC UGAGAGCUAC ACGCUGACGC CGGAGGUGGG 2100
GGAGCUCAUU GAGAAGGUGC GCAAAGCGCA CCAGGAAACC UUCCCUGCCC UCUGCCAGCU 2160
GGGCAAAUAC ACUACGAACA ACAGCUCAGA ACAACGUGUC UCUCUGGACA UUGACCUCUG 2220
GGACAAGUUC AGUGAACUCU CCACCAAGUG CAUCAUUAAG ACUGUGGAGU UCGCCAAGCA 2280
GCUGCCCGGC UUCACCACCC UCACCAUCGC CGACCAGAUC ACCCUCCUCA AGGCUGCCUG 2340 CCUGGACAUC CUGAUCCUGC GGAUCUGCAC GCGGUACACG CCCGAGCAGG ACACCAUGAC 2 00
CUUCUCGGAC GGGCUGACCC UGAACCGGAC CCAGAUGCAC AACGCUGGCU UCGGCCCCCU 2460
CACCGACCUG GUCUUUGCCU UCGCCAACCA GCUGCUGCCC CUGGAGAUGG AUGAUGCGGA 2520
GACGGGGCUG CUCAGCGCCA UCUGCCUCAU CUGCGGAGAC CGCCAGGACC UGGAGCAGCC 2580
GGACCGGGUG GACAUGCUGC AGGAGCCGCU GCUGGAGGCG CUAAAGGUCU ACGUGCGGAA 2640
GCGGAGGCCC AGCCGCCCCC ACAUGUUCCC CAAGAUGCUA AUGAAGAUUA CUGACCUGCG 2700
AAGCAUCAGC GCCAAGGGGG CUGAGCGGGU GAUCACGCUG AAGAUGGAGA UCCCGGGCUC 2760
CAUGCCGCCU CUCAUCCAGG AAAUGUUGGA GAACUCAGAG GGCCUGGACA CUCUGAGCGG 2820
ACAGCCGGGG GGUGGGGGGC GGGACGGGGG UGGCCUGGCC CCCCCGCCAG GCAGCUGUAG 2880
CCCCAGCCUC AGCCCCAGCU CCAACAGAAG CAGCCCGGCC ACCCACUCCC CGUGACCGCC 2940
CACGCCACAU GGACACAGCC CUCGCCCUCC GCCCCGGCUU UUCUCUGCCU UUCUACCGAC 3000
CAUGUGACCC CGCACCAGCC CUGCCCCCAC CUGCCCUCCC GGGCAGUACU GGGGACCUUC 3060
CCUGGGGGAC GGGGAGGGAG GAGGCAGCGA CUCCUUGGAC AGAGGCCUGG GCCCUCAGUG 3120
GACUGCCUGC UCCCACAGCC UGGGCUGACG UCAGAGGCCG AGGCCAGGAA CUGAGUGAGG 3180
CCCCUGGUCC UGGGUCUCAG GAUGGGUCCU GGGGGCCUCG UGUUCAUCAA GACACCCCUC 3240
UGCCCAGCUC ACCACAUCUU CAUCACCAGC AAACGCCAGG ACUUGGCUCC CCCAUCCUCA 3300
GAACUCACAA GCCAUUGCUC CCCAGCUGGG GAACCUCAAC CUCCCCCCUG CCUCGGUUGG 3360
UGACAGAGGG GGUGGGACAG GGGCGGGGGG UUCCCCCUGU ACAUACCCUG CCAUACCAAC 3420
CCCAGGUAUU AAUUCUCGCU GGUUUUGUUU UUAUUUUAAU UUUUUUGUUU UGAUUUUUUU 3480
AAUAAGAAUU UUCAUUUUAA GCAAAAAAAA A 3511
(2) INFORMATION FOR SEQ ID NO:4 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1481 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: RNA
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..1481
(D) OTHER INFORMATION: /function= "RARα RNA Sequence."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
GGGGGCGGGC ACCUCAAUGG GUACCCGGUG CCUCCCUACG CCUUCUUCUU CCCCCCUAUG 60
CUGGGUGGAC UCUCCCCGCC AGGCGCUCUG ACCACUCUCC AGCACCAGCU UCCAGUUAGU 120
GGAUAUAGCA CACCAUCCCC AGCCACCAUU GAGACCCAGA GCAGCAGUUC UGAAGAGAUA 180
GUGCCCAGCC CUCCCUCGCC ACCCCCUCUA CCCCGCAUCU ACAAGCCUUG CUUUGUCUGU 240 CAGGACAAGU CCUCAGGCUA CCACUAUGGG GUCAGCGCCU GUGAGGGCUG CAAGGGCUUC 300
UUCCGCCGCA GCAUCCAGAA GAACAUGGUG UACACGUGUC ACCGGGACAA GAACUGCAUC 360
AUCAACAAGG UGACCCGGAA CCGCUGCCAG UACUGCCGAC UGCAGAAGUG CUUUGAAGUG 420
GGCAUGUCCA AGGAGUCUGU GAGAAACGAC CGAAACAAGA AGAAGAAGGA GGUGCCCAAG 480
CCCGAGUGCU CUGAGAGCUA CACCGUGACG CCGGAGGUGG GGGAGCUCAU UGAGAAGGUG 540
CGUAAAGCGC ACCAGGAAAC CUUCCCUGCC CUCUGCCAGC UGGGCAAAUC AAUCACGAAC 600
AACAGCUCAG AACAACGUGU CUCUCUGGAC AUUGACCUCU GGGACAAGUU CAGUGAACUC 660
UCCACCAAGU GCAUCAUUAA GACUGUGGAG UUCGCCAAGC AGCUGCCCGG CUUCACCACC 720
CUCACCAUCG CCGACCAGAU CACCCUCCUC AAGGCUGCCU GCCUGGACAU CCUGAUCCUG 780
CGGAUCUGCA CGCGGUACAC GCCCGAGCAG GACACCAUGA CCUUCUCGGA CGGGCUGACC 840
CUGAACCGGA CCCAGAUGCA CAACGCUGGC UUCGGCCCCC UCACCGACCU GGUCUUUGCC 900
UUCGCCAACC AGGACCGGGU GGACAUGCUG CAGGAGCCGC UGCUGGAGGC GCUAAAGGUC 960
UACGUGCGGA AGCGGAGGCC CAGCCGCCCC CACAUGUUCC CCAAGAUGCU AAUGAAGAUU 1020
ACUGACCUGC GAAGCAUCAG CGCCAACGGG GCUGAGCGGG UGAUCACGCU GAAGAUGGAG 1080
AUCCCGGGCU CCAGUCCGCC UCUCAUCCAG GAAAUGUUGG AGAACUCAGA GGGCCUGGAC 1140
ACUCUGAGCG GACAGCCGGG GGGUGGGGGG CGGGACGGGG GUCGCCACAU GGACACAGCC 1200
CUGGCCCUCC GCCCCCGGCU UUUCUCUGCC UUUCUACCGA CCAUGUGACC CCGCACCAGC 1260
CCUGCCCCCA CCUGCCCUGC CCGGGAGUAC UGGCGACCUU CCCUGGGGGA CGGGGAGGGA 1320
GGAGGCAGCG ACUCCUUGGA CAGAGGCCUG GGCCCUCAGU GGACUGCCUG CUCCCACAGC 1380
CUGGGCUGAC GUCAGAGGCC GAGGCCAGGA ACUGAGUGAG GCCCCUGGUC CUGGGUCUCA 1440
GGAUGGGUCC UGGGGGCCUC GUGUUCAUCA AGACGGAAUU C 1481
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: RNA
(iii) HYPOTHETICAL: NO
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..36
(D) OTHER INFORMATION: /function="Ribozyme construct IHRZl.8" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5 :
GGUCUCCUGA UGAGUCCGUG AGGACGAAAU GGCUGC 36
(2) INFORMATION FOR SEQ ID NO:6 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: RNA (iii) HYPOTHETICAL: NO
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..41
(D) OTHER INFORMATION: /function="Ribozyme construct IHRZl.3" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6 :
CUUCACUGAU GAGUCCGUGA GGACGAAAAC CCCAUGGCUG C 41
(2) INFORMATION FOR SEQ ID NO:7 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: RNA
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..42
(D) OTHER INFORMATION: /function-*"Ribozyme construct IHRZl.30"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
GGUCUCCUGA UGAGUCCGUG AGGACGAAAU GCCCACUGGC CA 42
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8 :
TGGGTCTCAA TGGCTGCCTC CCCGG 25
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9 :
GTCTCAATGG CTGCCTCCCC GGCGC 25
(2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: CTCTGGGTCT CAATGGCTGC CTCCC 25
(2) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
CCGGGGAGGC AGCCATTGAG ACCCA 25
(2) INFORMATION FOR SEQ ID NO:12 : (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 51 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: RNA
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..51
(D) OTHER INFORMATION: /function="anti-APL construct 1.1 (hammerhead ribozyme) "
(ix) FEATURE:
(A) NAME/KEY: variant
(B) LOCATION: 17; 36
(D) OTHER INFORMATION: /function="variant (5.1) delete G at position 17 and replace A with C at position 36"
(ix) FEATURE:
(A) NAME/KEY: variant
(B) LOCATION: 1...8; 47...51
(D) OTHER INFORMATION: /function="variant (1.0) delete residues at 1-8 and 47-51"
(ix) FEATURE:
(A) NAME/KEY: variant
(B) LOCATION: 1...8; 17; 36; 47...51
(D) OTHER INFORMATION: /function-*"variant (5.0) delete residues at 1-8 and 47-51 and delete G at position 17 and replace A with C at position 36"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
CUGCUCUGGG UCUCCUGAUG AGUCCGUGAG GACGAAAUGG CUGCCUCCCC G 51
(2) INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: RNA
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..50
(D) OTHER INFORMATION: /function="APL RNA" ( ix) FEATURE :
(A) NAME/KEY: misc_feature
(B) LOCATION: 11; 12
(D) OTHER INFORMATION: /function--"fusion junction"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
CGGGGAGGCA GCCAUUGAGA CCCAGAGCAG CAGUUCUGAA GAGAUAGUGC 50
(2) INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 55 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: RNA
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..55
(D) OTHER INFORMATION: /function="anti-APL construct 2.1 (hammerhead ribozyme) "
(ix) FEATURE:
(A) NAME/KEY: variant
(B) LOCATION: 17; 36
(D) OTHER INFORMATION: /function="variant (6.1) delete G at position 17 and replace A with C at position 36"
(ix) FEATURE:
(A) NAME/KEY: variant
(B) LOCATION: 1...9; 51...55
(D) OTHER INFORMATION: /function="variant (2.0) delete residues at 1-9 and 51-55"
(ix) FEATURE:
(A) NAME/KEY: variant
(B) LOCATION: 1...9; 17; 36; 51...55
(D) OTHER INFORMATION: /function="variant (6.0) delete residues at 1-9 and 51-55 and delete G at position 17 and replace A with C at position 36"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
GCACUAUCUC UUCACUGAUG AGUCCGUGAG GACGAAAACC CCAUGGCUGC CUCCC 55
(2) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: RNA
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..35
(D) OTHER INFORMATION: /function="APL EGS A20"
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 22...23
(D) OTHER INFORMATION: /function="variant (A20D) delete U and U at positions 22 and 23" (ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 17...23
(D) OTHER INFORMATION: /function="sequence at 17-23 is phosphorothioate RNA; remainder of the molecule is composed of 2 ' -O methyl RNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
GGGUCUCAGG CCCGGGUUCG AUUCCCGGUG GCUGC 35
(2) INFORMATION FOR SEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: RNA
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..31
(D) OTHER INFORMATION: /function="APL EGS 1009"
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 14; 17; 18; 29
(D) OTHER INFORMATION: /function="variant (1017) delete RNA at positions 14, 17, 18 and 29 (U, A, A, and G, respectively) "
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 13...19
(D) OTHER INFORMATION: /function="sequence at 13-19 is phosphorothioate RNA; remainder of the molecule is composed of 2'-0 methyl RNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
GUCUCAAGAA GGUUCGAAUC CUUCGGCUGC C 31

Claims

We claim :
1. A RNA molecule comprising nucleotide guide sequences complementary to specific sequences of a defined cancer- associated RNA molecule, wherein the nucleotide guide sequences base pair to the specific cancer-associated RNA to form a hybrid structure which promotes ribozyme-mediated cleavage of the cancer-associated RNA.
2. The RNA molecule of claim 1 wherein the cancer- associated RNA is selected from the group consisting of acute promyelocytic cancer-associated RNA PML-RARα, follicular lymphoma-associated RNA, and chronic myelocytic leukemia- associated RNA.
3. The RNA molecule of claim 1 selected from the group consisting of hammerhead ribozymes, hairpin ribozymes, hepatitis delta virus-derived ribozymes, and external guide sequences for RNase P.
4. The RNA molecule of claim 1 wherein the RNA is modified to include a chemical modification selected from the group consisting of modification of the phosphodiester bond to methyl phosphonate bond; modification of the phosphodiester bond to phosphorothioate bond; substitution of 2' hydroxyl group of the ribonucleotides with hydrogen, with a methoxy group or other 0-alkyl group, with an amino group, or with fluorine, wherein the modification increases resistance of the RNA to nucleases.
5. The RNA molecule of claim 1 wherein the oligonucleotide is chemically modified to increase resistance to nucleases, wherein the modification is selected from the group consisting of modification of the phosphodiester bond to methylphosphonate or phosphorothioate and substitution of the 2' position of the ribose with methoxy, O-alkyl, amino or fluoro group.
6. A method for inhibiting a cancer comprising administering to a patient or cells from a patient an RNA molecule which is in a pharmaceutically acceptable delivery system and which comprises nucleotide guide sequences which hybridize to and promote ribozyme-mediated cleavage of a specific cancer-associated RNA.
7. The method of claim 6 wherein the cancer is selected from the group consisting of acute promyelocytic leukemia, follicular lymphoma and chronic myelocytic leukemia.
8. The method of claim 6 wherein the RNA molecule is selected from the group consisting of hammerhead ribozymes, hairpin ribozymes, hepatitis delta virus-derived ribozymes, and external guide sequences to RNase P.
9. The method of claim 6 wherein the RNA is modified to include a chemical modification selected from the group consisting of modification of the phosphodiester bond to methyl phosphonate bond; modification of the phosphodiester bond to phosphorothioate bond; substitution of 2' hydroxyl group of the ribonucleotides with hydrogen, with a methoxy group or other O-alkyl group, with an amino group, or with fluorine, wherein the modification increases resistance of the RNA to nucleases.
10. The method of claim 6 wherein the RNA molecule is generated by transcription of a DNA molecule encoding the RNA molecule.
11. The method of claim 10 wherein the DNA molecule is in a liposome-complexed plasmid.
12. The method of claim 10 wherein the DNA molecule is in a viral vector selected from the group consisting of retroviral vectors, adeno-associated virus-derived vectors, and Epstein-Barr virus-derived vectors.
13. A method for screening the ability of oligonucleotides with ribozyme or EGS properties to cleave and inactivate a cancer-associated mRNA comprising the steps of
(i) synthesizing a cancer-associated mRNA and its natural, non-cancerous counterpart;
(ii) reacting in vi tro the mRNAs with the oligonucleotides to be screened; and
(iii) determining the cleavage activity of the oligonucleotides being screened.
1 . A DNA oligonucleotide molecule complementary to the fusion junction of the PML-RAR mRNA, binding to the fusion junction and promoting Rnase H cleavage of the mRNA or translation blockage of the PML-RAR mRNA.
15. The oligonucleotide of claim 14 wherein the oligonucleotide is chemically modified to increase resistance to nucleases, wherein the modification is selected from the group consisting of modification of the phosphodiester bond to methylphosphonate or phosphorothioate and substitution of the 2' position of the ribose with methoxy, O-alkyl, amino or fluoro group.
16. The oligonucleotide of claim 14 wherein the affinity of the oligonucleotide for the APL RNA is increased by the addition of a propyne moiety to the C5 carbon of the bases.
17. The oligonucleotide of claim 14 wherein the oligonucleotide is complexed with liposomes.
18. The oligonucleotide of claim 14 wherein the oligonucleotide in its RNA form is expressed in the APL cells by an engineered expression vector under the control of an RNA polymerase II or RNA polymerase III promoter.
19. The oligonucleotide of claim 18 wherein the vector is a liposome complexed plasmid.
20. The oligonucleotide of claim 18 wherein the vector is a viral vector selected from the group consisting of retroviral vectors, adeno-associated viral vectors and Epstein-Barr viral vectors.
21. A method for inhibiting a leukemia comprising administering to a patient or cells from a patient a DNA oligonucleotide molecule complementary to the fusion junction of the PML-RAR mRNA, binding to the fusion junction and promoting Rnase H cleavage of the mRNA or translation blockage of the PML-RAR mRNA.
22. The method of claim 21 wherein the oligonucleotide is chemically modified to increase resistance to nucleases, wherein the modification is selected from the group consisting of modification of the phosphodiester bond to methylphosphonate or phosphorothioate and substitution of the 2' position of the ribose with methoxy, O-alkyl, amino or fluoro group.
23. The method of claim 21 wherein the affinity of the oligonucleotide for the APL RNA is increased by the addition of a propyne moiety to the C5 carbon of the bases.
24. The method of claim 21 wherein the oligonucleotide is complexed with liposomes.
25. The method of claim 21 wherein the oligonucleotide in its RNA form is expressed in the APL cells by an engineered expression vector under the control of an RNA polymerase II or RNA polymerase III promoter.
26. The method of claim 25 wherein the vector is a liposome complexed plasmid.
27. The method of claim 25 wherein the vector is a viral vector selected from the group consisting of retroviral vectors, adeno-associated viral vectors and Epstein-Barr viral vectors.
PCT/US1995/016451 1994-12-14 1995-12-14 Ribozyme-mediated inactivation of leukemia-associated rna WO1996018733A2 (en)

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