WO1996018733A2 - Inactivation induite par ribozyme de l'arn associe a la leucemie - Google Patents

Inactivation induite par ribozyme de l'arn associe a la leucemie 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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WO1996018733A3 (fr
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Umberto Pace
Shaji T. George
Allan R. Goldberg
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Innovir Laboratories, Inc.
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Publication of WO1996018733A3 publication Critical patent/WO1996018733A3/fr

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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

La présente invention concerne la construction de molécules d'ARN, y compris de ribozymes, de séquences guides externes pour la ribonucléase P et d'oligonucléotides antisens, lesquelles molécules, suivant le cas, favorisent la coupure par ribozymes d'ARN spécifiques de cancers, ou en bloquent la transcription. Ces ARN sont notamment l'ARN de la leucémie promyéloleucocytaire aiguë, l'ARN du lymphome folliculaire, et l'ARN de la leucémie myéloloïde chronique. L'invention concerne également des procédés de production et d'utilisation de telles molécules d'ARN.
PCT/US1995/016451 1994-12-14 1995-12-14 Inactivation induite par ribozyme de l'arn associe a la leucemie WO1996018733A2 (fr)

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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996021731A2 (fr) * 1995-01-13 1996-07-18 Innovir Laboratories, Inc. Sequences guides externes stabilisees
WO1997018305A2 (fr) * 1995-11-14 1997-05-22 Regents Of The University Of Minnesota Procede pour preparer des cellules hematopoietiques non malignes pharmacoresistantes
WO1997033991A1 (fr) * 1996-03-14 1997-09-18 Innovir Laboratories, Inc. Sequence guides externes courtes
WO1998024928A2 (fr) * 1996-12-06 1998-06-11 Niels Pallisgaard Detection d'anomalies chromosomiques
US5869248A (en) * 1994-03-07 1999-02-09 Yale University Targeted cleavage of RNA using ribonuclease P targeting and cleavage sequences
WO1999027135A2 (fr) * 1997-11-21 1999-06-03 Yale University Procede d'identification et d'inhibition de molecules fonctionnelles d'acide nucleique dans des cellules
US5976874A (en) * 1996-08-16 1999-11-02 Yale University Phenotypic conversion of drug-resistant bacteria to drug-sensitivity
US6013447A (en) * 1997-11-21 2000-01-11 Innovir Laboratories, Inc. Random intracellular method for obtaining optimally active nucleic acid molecules
US6057153A (en) * 1995-01-13 2000-05-02 Yale University Stabilized external guide sequences
US6248525B1 (en) 1998-03-30 2001-06-19 Yale University Method for identifying essential or functional genes
FR2832154A1 (fr) * 2001-11-09 2003-05-16 Centre Nat Rech Scient Oligonucleotides inhibiteurs et leur utilisation pour reprimer specifiquement un gene
EP2241623A2 (fr) 1999-07-07 2010-10-20 ZymoGenetics, Inc. Anti-corps monoclonal contre un récepteur de cytokine humain
US8044032B2 (en) 2000-01-31 2011-10-25 Yissum Research Development Company Of The Hebrew University Of Jerusalem Selective killing of cells by activation of double-stranded RNA dependent protein kinase-PKR
US8673870B2 (en) * 2002-04-26 2014-03-18 Institut National De La Sante Et De La Recherche Medicale (Inserm) Combined DNA vaccine and biological modifiers for cancer therapy

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WO1993023057A1 (fr) * 1992-05-14 1993-11-25 Ribozyme Pharmaceuticals, Inc. Procede et reactif destines a empecher l'evolution du cancer
WO1995023225A2 (fr) * 1994-02-23 1995-08-31 Ribozyme Pharmaceuticals, Inc. Procede et reactif inhibiteur de l'expression de genes concernant une affection

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WO1995023225A2 (fr) * 1994-02-23 1995-08-31 Ribozyme Pharmaceuticals, Inc. Procede et reactif inhibiteur de l'expression de genes concernant une affection

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BLOOD 86 (10 SUPPL. 1). PAGE 263A, ABSTRACT 1037, 15 November 1995, XP002008611 NASON-BURCHENAL, K. ET AL.: "Over-expression of ribozymes cleaving PML-RAR-alpha in vitro is cytotoxic to acute promyelocytic leukemia cells in vivo." & 37TH ANNUAL MEETING OF THE AMERICAN SOCIETY OF HEMATOLOGY, SEATTLE, WASHINGTON, USA, DECEMBER 1-5, 1995., *
CANCER RESEARCH, (1994 DEC 15) 54 (24) 6365-9., XP002008609 PACE, U. ET AL.: "A ribozyme which discriminates in vitro between PML/RAR alpha, the t(15;17)-associated fusion RNA of acute promyelocytic leukemia, and PML and RAR alpha, the transcripts from the nonrearranged alleles." *
CELL, vol. 66, 23 August 1991, NA US, pages 675-684, XP002008608 DE THŸ, H. ET AL.: "The PML-RAR alpha fusion mRNA generated by the t(15;17) translocation in acute promylocytic leukemia encods a functionally altered RAR" cited in the application *
CURRENT OPINION IN STRUCTURAL BIOLOGY, vol. 4, no. 3, 1 June 1994, pages 322-330, XP000523747 SYMONS, R. H.: "RIBOZYMES" *
EMBO JOURNAL, vol. 11, no. 5, 1 May 1992, pages 1913-1919, XP000268586 PAOLELLA, G. ET AL.: "NUCLEASE RESISTANT RIBOZYMES WITH HIGH CATALYTIC ACTIVITY" cited in the application *
JOURNAL OF CELLULAR BIOCHEMISTRY SUPPLEMENT 0 (19A).1995. PAGE 223, ABSTRACT A6-317, XP002008610 NORDSTROM, J. ET AL.: "External guide sequences that direct human ribonuclease P to cleave the fusion junction of the PML-RAR-alpha RNA of acute promyelocytic leukemia." & KEYSTONE SYMPOSIUM ON RIBOZYMES: BASIC SCIENCE AND THERAPEUTIC APPLICATIONS, BRECKENRIDGE, COLORADO, USA, 15 - 21 January 1995, *
PROC ANNU MEET AM ASSOC CANCER RES, VOL. 35, PAGE 206, ABSTRACT #1227, March 1994, XP002008607 PACE, U. ET AL.: "A ribozyme which discriminates in vitro between the acute promyelocytic leukemia t(15;17) fusion RNA, PML/RAR alpha, and the normal transcripts from the non-rearranged alleles." & 85TH ANN.MEET. AM.ASSOC. CANCER RES.; SAN FRANCISCO, 10 - 13 April 1994, *
TRENDS IN BIOTECHNOLOGY, vol. 8, no. 7, 1 July 1990, pages 179-183, XP000133090 ROSSI, J. ET AL.: "RNA ENZYMES (RIBOZYMES) AS ANTIVIRAL THERAPEUTIC AGENTS" *

Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5869248A (en) * 1994-03-07 1999-02-09 Yale University Targeted cleavage of RNA using ribonuclease P targeting and cleavage sequences
WO1996021731A3 (fr) * 1995-01-13 1996-12-27 Innovir Lab Inc Sequences guides externes stabilisees
US5683873A (en) * 1995-01-13 1997-11-04 Innovir Laboratories, Inc. EGS-mediated inactivation of target RNA
WO1996021731A2 (fr) * 1995-01-13 1996-07-18 Innovir Laboratories, Inc. Sequences guides externes stabilisees
US6057153A (en) * 1995-01-13 2000-05-02 Yale University Stabilized external guide sequences
WO1997018305A2 (fr) * 1995-11-14 1997-05-22 Regents Of The University Of Minnesota Procede pour preparer des cellules hematopoietiques non malignes pharmacoresistantes
WO1997018305A3 (fr) * 1995-11-14 1997-07-10 Univ Minnesota Procede pour preparer des cellules hematopoietiques non malignes pharmacoresistantes
WO1997033991A1 (fr) * 1996-03-14 1997-09-18 Innovir Laboratories, Inc. Sequence guides externes courtes
US5877162A (en) * 1996-03-14 1999-03-02 Innovir Laboratories, Inc. Short external guide sequences
US5976874A (en) * 1996-08-16 1999-11-02 Yale University Phenotypic conversion of drug-resistant bacteria to drug-sensitivity
WO1998024928A2 (fr) * 1996-12-06 1998-06-11 Niels Pallisgaard Detection d'anomalies chromosomiques
WO1998024928A3 (fr) * 1996-12-06 1999-03-25 Niels Pallisgaard Detection d'anomalies chromosomiques
WO1999027135A2 (fr) * 1997-11-21 1999-06-03 Yale University Procede d'identification et d'inhibition de molecules fonctionnelles d'acide nucleique dans des cellules
US6013447A (en) * 1997-11-21 2000-01-11 Innovir Laboratories, Inc. Random intracellular method for obtaining optimally active nucleic acid molecules
WO1999027135A3 (fr) * 1997-11-21 1999-07-29 Innovir Lab Inc Procede d'identification et d'inhibition de molecules fonctionnelles d'acide nucleique dans des cellules
US6248525B1 (en) 1998-03-30 2001-06-19 Yale University Method for identifying essential or functional genes
EP2241623A2 (fr) 1999-07-07 2010-10-20 ZymoGenetics, Inc. Anti-corps monoclonal contre un récepteur de cytokine humain
US8044032B2 (en) 2000-01-31 2011-10-25 Yissum Research Development Company Of The Hebrew University Of Jerusalem Selective killing of cells by activation of double-stranded RNA dependent protein kinase-PKR
FR2832154A1 (fr) * 2001-11-09 2003-05-16 Centre Nat Rech Scient Oligonucleotides inhibiteurs et leur utilisation pour reprimer specifiquement un gene
US8318689B2 (en) 2001-11-09 2012-11-27 Centre National De La Recherche Scientifique SiRNA-based cancer treatment
US8673870B2 (en) * 2002-04-26 2014-03-18 Institut National De La Sante Et De La Recherche Medicale (Inserm) Combined DNA vaccine and biological modifiers for cancer therapy

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