US 20050176038 A1
A transcribed non-naturally occurring RNA molecule comprising a desired 10 RNA molecule, wherein the 3+ region of the RNA is able to base-pair with at least 8 bases at the 5′ terminus of the same RNA molecule.
1. A transcribed non-naturally occurring RNA molecule having hairpin structure, comprising a desired RNA portion, wherein said desired RNA portion is present between a 3′ region and 5′ region, wherein said 3′ region and said 5′ region form an intramolecular stem with each other comprising at least 8 base pairs.
2. The RNA molecule of
3. The RNA molecule of
4. The RNA molecule of
5. The RNA molecule of
6. The RNA molecule of
7. The RNA molecule of
8. The RNA molecule of
9. The RNA molecule of
10. The RNA molecule of
11. The RNA molecule of
12. A DNA vector encoding the RNA molecule of
13. A RNA vector encoding the RNA molecule of
14. The DNA vector of
15. A cell comprising the vector of
16. A cell comprising the vector of
17. A cell comprising the RNA of
18. A method to provide a desired first RNA molecule having hairpin structure in a cell, comprising introducing into said cell a second RNA molecule comprising a 5′ region, a 3′ region, and said desired first RNA molecule, wherein said 5′ terminus is able to base pair with at least 8 bases of said 3′ region, and wherein said desired first RNA molecule is present between the bases of the 3′ region and the 5′ terminus capable of base pairing in the second RNA molecule under conditions suitable to provide the desired first RNA molecule in the cell.
19. The method of
20. The RNA molecule of
21. The RNA molecule of
22. The RNA molecule of
23. The RNA molecule of
24. The RNA molecule of
25. The RNA molecule of
This application is a continuation-in-part of James Thompson, “Improved RNA Polymerase III-Based Expression of Therapeutic RNAS”, U.S. Ser. No. 08/293,520, filed Aug. 19, 1994, and James Thompson, “Improved RNA Polymerase III-Based Expression of Therapeutic RNAS”, U.S. Ser. No. 08/337,608, filed Nov. 10, 1994, hereby incorporated by reference herein.
This invention relates to RNA polymerase III-based methods and systems for expression of therapeutic RNAs in cells in vivo or in vitro.
The RNA polymerase III (pol III) promoter is one found in DNA encoding 5S, U6, adenovirus VA1, Vault, telomerase RNA, tRNA genes, etc., and is transcribed by RNA polymerase III (for a review see Geiduschek and Tocchini-Valentini, 1988 Annu. Rev. Biochem. 57, 873-914; Willis, 1993 Eur. J. Biochem. 212, 1-11). There are three major types of pol III promoters: types 1, 2 and 3 (Geiduschek and Tocchini-Valentini, 1988 supra; Willis, 1993 supra) (see
The type 2 pol III promoter is characterized by the presence of two cis-acting sequence elements downstream of the transcription start site. All Transfer RNA (tRNA), adenovirus VA RNA and Vault RNA (Kikhoefer et al., 1993, J. Biol. Chem. 268, 7868-7873) genes are transcribed using this promoter (Geiduschek and Tocchini-Valentini, 1988 supra; Willis, 1993 supra). The sequence composition and orientation of the two cis-acting sequence elements—A box (5′ sequence element) and B box (3′ sequence element) are essential for optimal transcription by RNA polymerase III.
The type 3 pol III promoter contains all of the cis-acting promoter elements upstream of the transcription start site. Upstream sequence elements include a traditional TATA box (Mattaj et al., 1988 Cell 55, 435-442), proximal sequence element (PSE) and a distal sequence element (DSE; Gupta and Reddy, 1991 Nucleic Acids Res. 19, 2073-2075). Examples of genes under the control of the type 3 pol III promoter are U6 small nuclear RNA (U6 snRNA) and Telomerase RNA genes.
In addition to the three predominant types of pol III promoters described above, several other pol III promoter elements have been reported (Willis, 1993 supra) (see
Gilboa WO 89/11539 and Gilboa and Sullenger WO 90/13641 describe transformation of eucaryotic cells with DNA under the control of a pol III promoter. They state:
The authors describe a fusion product of a chimeric tRNA and an RNA product (see
Adeniyi-Jones et al.,1984 Nucleic Acids Res. 12, 1101-1115, describe certain constructions which “may serve as the basis for utilizing the tRNA gene as a ‘portable promoter’ in engineered genetic constructions.” The authors describe the production of a so-called Δ3′-5 in which 11 nucleotides of the 3′-end of the mature tRNAi met sequence are replaced by a plasmid sequence, and are not processed to generate a mature tRNA. The authors state:
Sullenger et al., 1990 Cell 63, 601-619, describe over-expression of TAR-containing sequences using a chimeric tRNAi met-TAR transcription unit in a double copy (DC) murine retroviral vector.
Sullenger et al., 1990 Molecular and Cellular Bio. 10, 6512, describe expression of chimeric tRNA driven antisense transcripts. It indicates:
Cotten and Birnstiel,1989 EMBO Jrnl. 8, 3861, describe the use of tRNA genes to increase intracellular levels of ribozymes. The authors indicate that the ribozyme coding sequences were placed between the A and the B box internal promoter sequences of the Xenopus tRNAmet gene. They also indicate that the targeted hammerhead ribozymes were active in vivo.
Yu et al., 1993 Proc. Natl. Acad. Sci. USA 90, 5340, describe the use of a VAI promoter to express a hairpin ribozyme. The resulting transcript consisted of the first 104 nucleotides of the VAI RNA, followed by the ribozyme sequence and the terminator sequence.
Lieber and Strauss, 1995 Mol. Cellular Bio. 15, 540, inserted a hammerhead ribozyme sequence in the central domain of a VAI RNA.
Applicant has determined that the level of production of a foreign RNA, using a RNA polymerase III (pol III) based system, can be significantly enhanced by ensuring that the RNA is produced with the 5′ terminus and a 3′ region of the RNA molecule base-paired together to form a stable intramolecular stem structure. This stem structure is formed by hydrogen bond interactions (either Watson-Crick or non-Watson-Crick) between nucleotides in the 3′ region (at least 8 bases) and complementary nucleotides in the 5′ terminus of the same RNA molecule.
Although the example provided below involves a type 2 pol III gene unit, a number of other pol III promoter systems can also be used, for example, tRNA (Hall et al., 1982 Cell 29, 3-5), 5S RNA (Nielsen et al., 1993, Nucleic Acids Res. 21, 3631-3636), adenovirus VA RNA (Fowlkes and Shenk, 1980 Cell 22, 405-413), U6 snRNA (Gupta and Reddy, 1990 Nucleic Acids Res. 19, 2073-2075), vault RNA (Kickoefer et al., 1993 J. Biol. Chem. 268, 7868-7873), telomerase RNA (Romero and Blackburn, 1991 Cell 67, 343-353), and others.
The construct described in this invention is able to accumulate RNA to a significantly higher level than other constructs, even those in which 5′ and 3′ ends are involved in hairpin loops. Using such a construct the level of expression of a foreign RNA can be increased to between 20,000 and 50,000 copies per cell. This makes such constructs, and the vectors encoding such constructs, excellent for use in decoy, therapeutic editing and antisense protocols as well as for ribozyme formation. In addition, the molecules can be used as agonist or antagonist RNAs (affinity RNAS). Generally, applicant believes that the intramolecular base-paired interaction between the 5′ terminus and the 3′ region of the RNA should be in a double-stranded structure in order to achieve enhanced RNA accumulation.
Thus, in one preferred embodiment the invention features a pol III promoter system (e.g., a type 2 system) used to synthesize a chimeric RNA molecule which includes tRNA sequences and a desired RNA (e.g., a tRNA-based molecule).
The following exemplifies this invention with a type 2 pol III promoter and a tRNA gene. Specifically to illustrate the broad invention, the RNA molecule in the following example has an A box and a B box of the type 2 pol III promoter system and has a 5′ terminus or region able to base-pair with at least 8 bases of a complementary 3′ end or region of the same RNA molecule. This is meant to be a specific example. Those in the art will recognize that this is but one example, and other embodiments can be readily generated using other pol III promoter systems and techniques generally known in the art.
By “terminus” is meant the terminal bases of an RNA molecule, ending in a 3′ hydroxyl or 5′ phosphate or 5′ cap moiety. By “region” is meant a stretch of bases 5′ or 3′ from the terminus that are involved in base-paired interactions. It need not be adjacent to the end of the RNA. Applicant has determined that base pairing of at least one end of the RNA molecule with a region not more than about 50 bases, and preferably only 20 bases, from the other end of the molecule provides a useful molecule able to be expressed at high levels.
By “3′ region” is meant a stretch of bases 3′ from the terminus that are involved in intramolecular base-paired interaction with complementary nucleotides in the 5′ terminus of the same molecule. The 3′ region can be designed to include the 3′ terminus. The 3′ region therefore is ≧0 nucleotides from the 3′ terminus. For example, in the S35 construct described in the present invention (
By “tRNA molecule” is meant a type 2 pol III driven RNA molecule that is generally derived from any recognized tRNA gene. Those in the art will recognize that DNA encoding such molecules is readily available and can be modified as desired to alter one or more bases within the DNA encoding the RNA molecule and/or the promoter system. Generally, but not always, such molecules include an A box and a B box that consist of sequences which are well known in the art (and examples of which can be found throughout the literature). These A and B boxes have a certain consensus sequence which is essential for a optimal pol III transcription.
By “chimeric tRNA molecule” is meant a RNA molecule that includes a pol III promoter (type 2) region. A chimeric tRNA molecule, for example, might contain an intramolecular base-paired structure between the 3′ region and complementary 5′ terminus of the molecule, and includes a foreign RNA sequence at any location within the molecule which does not affect the activity of the type 2 pol III promoter boxes. Thus, such a foreign RNA may be provided at the 3′ end of the B box, or may be provided in between the A and the B box, with the B box moved to an appropriate location either within the foreign RNA or another location such that it is effective to provide pol III transcription. In one example, the RNA molecule may include a hammerhead ribozyme with the B box of a type 2 pol III promoter provided in stem II of the ribozyme. In a second example, the B box may be provided in stem IV region of a hairpin ribozyme. A specific example of such RNA molecules is provided below. Those in the art will recognize that this is but one example, and other embodiments can be readily generated using techniques generally known in the art.
By “desired RNA” molecule is meant any foreign RNA molecule which is useful from a therapeutic, diagnostic, or other viewpoint. Such molecules include antisense RNA molecules, decoy RNA molecules, enzymatic RNA, therapeutic editing RNA (Woolf and Stinchcomb, “Oligomer directed In situ reversion (ISR) of genetic mutations”, filed Jul. 6, 1994, U.S. Ser. No. 08/271,280, hereby incorporated by reference) and agonist and antagonist RNA.
By “antisense RNA” is meant a non-enzymatic RNA molecule that binds to another RNA (target RNA) by means of RNA-RNA interactions and alters the activity of the target RNA (Eguchi et al., 1991 Annu. Rev. Biochem. 60, 631-652). By “enzymatic RNA” is meant an RNA molecule with enzymatic activity (Cech, 1988 J. American. Med. Assoc. 260, 3030-3035). Enzymatic nucleic acids (ribozymes) act by first binding to a target RNA. Such binding occurs through the target binding portion of a enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA.
By “decoy RNA” is meant an RNA molecule that mimics the natural binding domain for a ligand. The decoy RNA therefore competes with natural binding target for the binding of a specific ligand. For example, it has been shown that over-expression of HIV trans-activation response (TAR) RNA can act as a “decoy” and efficiently binds HIV tat protein, thereby preventing it from binding to TAR sequences encoded in the HIV RNA (Sullenger et al., 1990 Cell 63, 601-608). This is meant to be a specific example. Those in the art will recognize that this is but one example, and other embodiments can be readily generated using techniques generally known in the art.
By “therapeutic editing RNA” is meant an antisense RNA that can bind to its cellular target (RNA or DNA) and mediate the modification of a specific base (Woolf and Stinchcomb, supra).
By “agonist RNA” is meant an RNA molecule that can bind to protein receptors with high affinity and cause the stimulation of specific cellular pathways.
By “antagonist RNA” is meant an RNA molecule that can bind to cellular proteins and prevent it from performing its normal biological function (for example, see Tsai et al., 1992 Proc. Natl. Acad. Sci. USA 89, 8864-8868).
By “complementary” is meant a RNA sequence that can form hydrogen bond(s) with other RNA sequence by either traditional Watson-Crick or other non-traditional types (for example, Hoogsteen type) of base-pairing interaction.
In other aspects, the invention includes vectors encoding RNA molecules as described above, cells including such vectors, methods for producing the desired RNA, and use of the vectors and cells to produce this RNA.
By “vectors” is meant any nucleic acid- and/or viral-based technique used to deliver a desired nucleic acid.
Thus, the invention features a transcribed non-naturally occuring RNA molecule which includes a desired therapeutic RNA portion and an intramolecular stem formed by base-pairing interactions between a 3′ region and complementary nucleotides at the 5′ terminus in the RNA. The stem preferably includes at least 8 base pairs, but may have more, for example, 15 or 16 base pairs.
In preferred embodiments, the 5′ terminus of the chimeric tRNA includes a portion of the precursor molecule of the primary tRNA molecule, of which ≧8 nucleotides are involved in base-pairing interaction with the 3′ region; the chimeric tRNA contains A and B boxes; natural sequences 3′ of the B box are deleted, which prevents endogenous RNA processing; the desired RNA molecule is at the 3′ end of the B box; the desired RNA molecule is between the A and the B box; the desired RNA molecule includes the B box; the desired RNA molecule is selected from the group consisting of antisense RNA, decoy RNA, therapeutic editing RNA, enzymatic RNA, agonist RNA and antagonist RNA; the molecule has an intramolecular stem resulting from a base-paired interaction between the 5′ terminus of the RNA and a complementary 3′ region within the same RNA, and includes at least 8 bases; and the 5′ terminus is able to base pair with at least 15 bases of the 3′ region.
In most preferred embodiments, the molecule is transcribed by a RNA polymerase III based promoter system, e.g., a type 2 pol III promoter system; the molecule is a chimeric tRNA, and may have the A and B boxes of a type 2 pol III promoter separated by between 0 and 300 bases; DNA vector encoding the RNA molecule of claim 1.
In other related aspects, the invention features an RNA or DNA vector encoding the above RNA molecule, with the portions of the vector encoding the RNA functioning as a RNA pol III promoter; or a cell containing the vector; or a method to provide a desired RNA molecule in a cell, by introducing the molecule into a cell with an RNA molecule as described above. The cells can be derived from animals, plants or human beings.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.
The drawings will first briefly be described.
To make internally-labeled substrate RNA for trans-ribozyme cleavage reactions, a 613 nt region (containing site I) was synthesized by PCR using primers that place the T7 RNA promoter upstream of the amplified sequence. Target RNA was transcribed, using T7 RNA polymerase, in a standard transcription buffer in the presence of [α-32P]CTP. The reaction mixture was treated with 15 units of ribonuclease-free DNasel, extracted with phenol followed chloroform:isoamyl alcohol (25:1), precipitated with isopropanol and washed with 70% ethanol. The dried pellet was resuspended in 20 μl DEPC-treated water and stored at −20° C.
Unlabeled ribozyme (200 nM) and internally labeled 613 nt substrate RNA (<10 nM) were denatured and renatured separately in a standard cleavage buffer (containing 50 mM Tris•HCl pH 7.5 and 10 mM MgCl2) by heating to 90° C. for 2 min. and slow cooling to 37° C. for 10 min. The reaction was initiated by mixing the ribozyme and substrate mixtures and incubating at 37° C. Aliquots of 5 μl were taken at regular time intervals, quenched by adding an equal volume of 2× formamide gel loading buffer and frozen on dry ice. The samples were resolved on 5% polyacrylamide sequencing gel and results were quantitatively analyzed by radioanalytic imaging of gels with a Phosphorlmager (Molecular Dynamics, Sunnyvale, Calif.).
Few antiviral drug therapies are available that effectively inhibit established viral infections. Consequently, prophylactic immunization has become the method of choice for protection against viral pathogens. However, effective vaccines for divergent viruses such as those causing the common cold, and HIV, the etiologic agent of AIDS, may not be feasible. Consequently, new antiviral strategies are being developed for combating viral infections.
Gene therapy represents a potential alternative strategy, where antiviral genes are stably transferred into susceptible cells. Such gene therapy approaches have been termed “intracellular immunization” since cells expressing antiviral genes become immune to viral infection (Baltimore, 1988 Nature 335, 395-396). Numerous forms of antiviral genes have been developed, including protein-based antivirals such as transdominant inhibitory proteins (Malim et al., 1993 J. Exp. Med., Bevec et al., 1992 P.N.A.S. (USA) 89, 9870-9874; Bahner et al., 1993 J. Virol. 67, 3199-3207) and viral-activated suicide genes (Ashorn et al., 1990 P.N.A.S. (USA) 87, 8889-8893). Although effective in tissue culture, protein-based antivirals have the potential to be immunogenic in vivo. It is therefore conceivable that treated cells expressing such foreign antiviral proteins will be eradicated by normal immune functions. Alternatives to protein based antivirals are RNA based molecules such as antisense RNAs, decoy RNAs, agonist RNAs, antagonist RNAs, therapeutic editing RNAs and ribozymes. RNA is not immunogenic; therefore, cells expressing such therapeutic RNAs are not susceptible to immune eradication.
In order for RNA-based gene therapy approaches to be effective, sufficient amounts of the therapeutic RNA must accumulate in the appropriate intracellular compartment of the treated cells. Accumulation is a function of both promoter strength of the antiviral gene, and the intracellular stability of the antiviral RNA. Both RNA polymerase II (pol II) and RNA polymerase III (pol III) based expression systems have been used to produce therapeutic RNAs in cells (Sarver & Rossi, 1993 AIDS Res. & Human Retroviruses 9, 483-487; Yu et al., 1993 P.N.A.S. (USA) 90, 6340-6344). However, pol III based expression cassettes are theoretically more attractive for use in expressing antiviral RNAs for the following reasons. Pol II produces messenger RNAs located exclusively in the cytoplasm, whereas pol III produces functional RNAs found in both the nucleus and the cytoplasm. Pol II promoters tend to be more tissue restricted, whereas pol III genes encode tRNAs and other functional RNAs necessary for basic “housekeeping” functions in all cell types. Therefore, pol III promoters are likely to be expressed in all tissue types. Finally, pol III transcripts from a given gene accumulate to much greater levels in cells relative to pol II genes.
Intracellular accumulation of therapeutic RNAs is also dependent on the method of gene transfer used. For example, the retroviral vectors presently used to accomplish stable gene transfer, integrate randomly into the genome of target cells. This random integration leads to varied expression of the transferred gene in individual cells comprising the bulk treated cell population. Therefore, for maximum effectiveness, the transferred gene must have the capacity to express therapeutic amounts of the antiviral RNA in the entire treated cell population, regardless of the integration site.
Pol III System
The following is just one non-limiting example of the invention. A pol III based genetic element derived from a human tRNAi met gene and termed Δ3-5 (
Applicant examined hammerhead (HHI) ribozyme (RNA with enzymatic activity) expression in human T cell lines using the Δ3-5 vector system (These constructs are termed “Δ3-5/HHI”;
The S35 gene unit may be used to express other therapeutic RNAs including, but not limited to, ribozymes, antisense, decoy, therapeutic editing, agonist and antagonist RNAs. Application of the S35 gene unit would not be limited to antiviral therapies, but also to other diseases, such as cancer, in which therapeutic RNAs may be effective. The S35 gene unit may be used in the context of other vector systems besides retroviral vectors, including but not limited to, other stable gene transfer systems such as adeno-associated virus (AAV; Carter, 1992 Curr. Opin. Genet. Dev. 3, 74), as well as transient vector systems such as plasmid delivery and adenoviral vectors (Berkner, 1988 BioTechniques 6, 616-629).
As described below, the S35 vector encodes a truncated version of a tRNA wherein the 3′ region of the RNA is base-paired to complementary nucleotides at the 5′ terminus, which includes the 5′ precursor portion that is normally processed off during tRNA maturation. Without being bound by any theory, Applicant believes this feature is important in the level of expression observed. Thus, those in the art can now design equivalent RNA molecules with such high expression levels. Below are provided examples of the methodology by which such vectors and tRNA molecules can be made.
The use of a truncated human tRNAi met gene, termed Δ3-5 (
Since the Δ3-5 vector combination has been successfully used to express inhibitory levels of both antisense and decoy RNAs, applicant cloned ribozyme-encoding sequences (termed as “Δ3-5/HHI”) into this vector to explore its utility for expressing therapeutic ribozymes. However, low ribozyme accumulation in human T cell lines stably transduced with this vector was observed (
Two strategies were used to try and protect the termini of the chimeric transcripts from exonucleolytic degredation. One strategy involved the incorporation of stem-loop structures into the termini of the transcript. Two such constructs were cloned, S3 which contains a stem-loop structure at the 3′ end, and S5 which contains stem-loop structures at both ends of the transcript (
Oligonucleotides encoding the S35 insert that overlap by at least 15 nucleotides were designed (5′ GATCCACTCTGCTGTTCTGTTTTTGA 3′ and 5′ CGCGTCAAAAACAGAACAGCAGAGTG 3′). The oligonucleotides (10 μM each) were denatured by boiling for 5 min in a buffer containing 40 mM Tris.HCl, pH 8.0. The oligonucleotides were allowed to anneal by snap cooling on ice for 10-15 min.
The annealed oligonucleotide mixture was converted into a double-stranded molecule using Sequenase® enzyme (US Biochemicals) in a buffer containing 40 mM Tris.HCl, pH 7.5, 20 mM MgCl2, 50 mM NaCl, 0.5 mM each of the four deoxyribonucleotide triphosphates, 10 mM DTT. The reaction was allowed to proceed at 37° C. for 30 min. The reaction was stopped by heating to 70° C. for 15 min.
The double stranded DNA was digested with appropriate restriction endonucleases (BamHI and MluI) to generate ends that were suitable for cloning into the Δ3-5 vector.
The double-stranded insert DNA was ligated to the Δ3-5 vector DNA by incubating at room temperature (about 20° C.) for 60 min in a buffer containing 66 mM Tris.HCl, pH 7.6, 6.6 mM MgCl2, 10 mM DTT, 0.066 μM ATP and 0.1U/μl T4 DNA Ligase (US Biochemicals).
Competent E. coli bacterial strain was transformed with the recombinant vector DNA by mixing the cells and DNA on ice for 60 min. The mixture was heat-shocked by heating to 37° C. for 1 min. The reaction mixture was diluted with LB media and the cells were allowed to recover for 60 min at 37° C. The cells were plated on LB agar plates and incubated at 37° C. for ˜18 h.
Plasmid DNA was isolated from an overnight culture of recombinant clones using standard protocols (Ausubel et al., Curr. Protocols Mol. Biology 1990, Wiley & Sons, New York).
The identity of the clones were determined by sequencing the plasmid DNA using the Sequenase® DNA sequencing kit (US Biochemicals).
The resulting recombinant Δ3-5 vector contains the S35 sequence. The HHI encoding DNA was cloned into this Δ3-5-S35 containing vector using SacII and BamHI restriction sites.
RNA from the transduced MT2 cells were extracted and the presence of Δ3-5/ribozyme chimeric transcripts were assayed by Northern analysis (Curr. Protocols Mol. Biol. 1992, ed. Ausubel et al., Wiley & Sons, New York). Northern analysis of RNA extracted from MT2 transductants showed that Δ3-5/ribozyme chimeras of appropriate sizes were expressed (
Addition of a stem-loop onto the 3′ end of Δ3-5/HHI did not lead to increased Δ3-5 levels (S3 in
Interestingly, the S35 construct expression in MT2 cells was about 100-fold more abundant relative to the original Δ3-5/HHI vector transcripts (
To assay whether ribozymes transcribed in the transduced cells contained cleavage activity, total RNA extracted from the transduced MT2 T cells were incubated with a labeled substrate containing the HHI cleavage site (
Variation in the ribozyme expression levels among cells making up the bulk population was determined by generating several clonal cell lines from the bulk S35 transduced CEM line (Curr. Protocols Mol. Biol. 1992, ed. Ausubel et al., Wiley & Sons, New York) and the ribozyme expression and activity levels in the individual clones were measured (
The fact that all 14 clones were found to express ribozyme indicate that the percentage of cells in the bulk population expressing ribozyme is also very high. In addition, the lowest level of expression in the clones was still more than 10-fold that seen in bulk cells transduced with the original Δ3-5 vector. Therefore, the S35 gene unit should be much more effective in a gene therapy setting in which bulk cells are removed, transduced and then reintroduced back into a patient.
Finally, the bulk S35-transduced line, resistant to G418, was propagated for a period of 3 months (in the absence of G418) to determine if ribozyme expression was stable over extended periods of time. This situation mimicks that found in the clinic in which bulk cells are transduced and then reintroduced into the patient and allowed to propogate. There was a modest 30% reduction of ribozyme expression after 3 months. This difference probably arose from cells with varying amount of ribozyme expression and exhibiting different growth rates in the culture becoming slightly more prevalent in the culture. However, ribozyme expression is apparently stable for at least this period of time.
A transcription unit, termed TRZ, is designed that contains the S35 motif (
Besides ribozymes, desired RNAs like antisense, therapeutic editing RNAs, decoys, can be readily inserted into the indicated region of TRZ-tRNA chimera to achieve therapeutic levels of RNA expression in mammalian cells.
A transcription unit, termed U6-S35, is designed that contains the characteristic intramolecular stem of a S35 motif (see
As a non-limiting example, applicant has constructed a stable, active ribozyme RNA driven from a eukaryotic U6 promoter (
By “unstructured” is meant lack of a secondary and tertiary structure such as lack of any stable base-paired structure within the sequence itself, and preferably with other sequences in the attached RNA.
By “spacer sequence” is meant any unstructured RNA sequence that separates the S35 domain from the desired RNA. The spacer sequence can be greater than or equal to one nucleotide.
In vitro Catalytic Activity of U6-S35-Ribozyme Chimeras
U6-S35-HHI ribozyme RNA was synthesized using T7 RNA polymerase. HHI RNA was chemically synthesized using RNA phosphoramidite chemistry as described in Wincott et al., 1995 Nucleic Acids Res. (in press). The ribozyme RNAs were gel-purified and the purified ribozyme RNAs were heated to 55° C. for 5 min. Target RNA used was ˜650 nucleotide long. Internally-32P-labeled target RNA was prepared as described above. The target RNA was pre-heated to 37° C. in 50 mM Tris.HCl, 10 mM MgCl2 and then mixed at time zero with the ribozyme RNAs (to give 200 nM final concentration of ribozyme). At appropriate times an aliquot was removed and the reaction was stopped by dilution in 95% formamide. Samples were resolved on a denaturing urea-polyacrylamide gel and products were quantitated on a phospholmager®.
As shown in
Accumulation of U6-S35-ribozyme Transcripts
An Actinomycin D assay was used to measure accumulation of the transcript in mammalian cells. Cells were transfected overnight with plasmids encoding the appropriate transcription units (2 μg bNA/well of 6 well plate) using calcium phosphate precipitation method (Maniatis et al., 1982 Molecular Cloning Cold Spring Harbor Laboratory Press, New York). After the overnight transfection, media was replaced and the cells were incubated an additional 24 hours. Cells were then incubated in media containing 5 μg/ml Actinomycin D. At the times indicated, cells were lysed in guanidinium isothiocyanate, and total RNA was purified by phenol/chloroform extraction and isopropanol precipitation as described by Chomczynski and Sacchi, 1987 Anal. Biochem., 162, 156. RNA was analyzed by northen blot analysis and the levels of specific RNAs were radioanalyticaly quantitated on a phospholmager®. The level of RNA at time zero was set to be 100%.
As shown in
Accumulation of VA1-S35-ribozyme Transcripts
An Actinomycin D assay was used to measure accumulation of the transcript in mammalian cells as described above. As shown in
Besides ribozymes, desired RNAs like antisense, therapeutic editing RNAs, decoys, can be readily inserted into the indicated U6-S35 or VA1-S35 chimera to achieve therapeutic levels of RNA expression in mammalian cells.
Sequences listed in the Figures are meant to be non-limiting examples. Those skilled in the art will recognize that variants (mutations, insertions and deletions) of the above examples can be readily generated using techniques known in the art, are within the scope of the present invention.
References cited herein, as well as Draper WO 93/23569, 94/02495, 94/06331, Sullenger WO 93/12657, Thompson WO 93/04573, and Sullivan WO 94/04609, and 93/11253 describe methods for use of vectors decribed herein, and are incorporated by reference herein. In particular these vectors are useful for administration of antisense and decoy RNA molecules.
Other embodiments are within the following claims.