AU2002246959B2 - Spliceosome mediated RNA trans-splicing - Google Patents

Description

18-02-'08 14:43 FEO- T-966 P008/099 F-541 00 SPLICEOSOME MEDIATED RNA TRANS-SPLICING 00

SPECIFICATION

The present application is a continuation-in-part of a pending application 09/838,858 filed on April 20, 2001 which is a continuation-in-part of pending application serial number 09/756096 filed January 8, 2001 which is a continuation-in-part of pending c-i application serial number 09/158,863 filed September 23, 1998 which is a continuation-in- 0 part of serial number 09/133,717 filed on August 13, 1998 which is a continuation-in-part of serial number 09/087,233 filed on May 28, 1998, which is a continuation-in-pant of pending application serial number 08/766,354 filed on December 13, 1996, which claims benefit to provisional application number 60/008,317 filed on December 15, 1995.

The present invention was made with government support under Grant Nos. SBIR R43DK56526-01 and SBIR R44DK56526-02. The government has certain rights in the invention.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:44 FROM- T-966 P009/099 F-541 00 02 0 1. INTRODUCTION 00 The present invention provides methods and compositions for generating novel nucleic acid molecules through targeted spliceosomal trans-splicing. The compositions of the invention include pre-trans-splicing molecules (PTMs) designed to interact with a natural target precursor messenger RNA molecule (target pre-mRNA) and mediate a transsplicing reaction resulting in the generation of a novel chimeric RNA molecule (chimeric RNA). The PTMs of the invention are genetically engineered so as to result in the cfI production of a novel chimeric NA which may itself perform a function, such as inhibiting the translation of the RNA, or that encodes a protein that complements a defective or. inactive protein in a cell, or encodes a toxin which kills specific cells.

Generally, the target pre-mRNA is chosen as a target because it is expressed within a specific cell type thus providing a means for targeting expression of the novel chimeric RNA to a selected cell type. The invention further relates to PTMs that have been genetically engineered for the COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:44 FROM- T-966 P010/0B99 F-541 00 0 0 0) identification of exon/intron boundaries of pre-mRNA molecules using an exon tagging method. In addition, PTMs can be designed to result in the production of 00 chimeric RNA encoding for peptide affinity purification tags which can be used to purify and identify proteins expressed in a specific cell type. The methods of the invention encompass contacting the PTMs of the invention with a target pre-mRNA under conditions in which a portion of the PTM is trans-spliced to a portion of the target pre-mRNA to form a novel ohiteric RNA molecule. The methods and N compositions of the invention can be used in cellular gene regulation, gene repair and Ssuicide gene therapy for treatment ofproliferative disorders such as cancer or C 10 treatment of genetic, autoimmune or infectious diseases. In addition, the methods and compositions of the invention can be used to generate novel nucleic acid molecules in plants through targeted splicesomal trans-splicing. For example, targeted trans-splicing may be used to regulate gene expression in plants for treatment ofplants diseases, engineering of disease resistant plants or expression of desirable genes in plants. The methods and compositions of the invention can also be used to map intron-exon boundaries and to identify novel proteins expressed in any given cell 2. BACKGROUND OF THE INVENTION DNA sequences in the chromosome are transcribed into pre-mRNAs which contain coding regions (exons) and generally also contain intervening noncoding regions (introns). Introns are removed from pre-nRNAs in a precise process called splicing (Chow et al, 1977, Cell 12:1-8; and Berget, S.M. et al., 1977, Proc.

Natl. Acad. Sci. USA 74:3171-3175). Splicing takes place as a coordinated interaction of several small nuclear ribonucleoprotein particles (snRNP's) and many protein factors that assemble to form an enzymatic complex known as the spliceosome (Moore et aL, 1993, in The RNA World, RF. Gestland and J.F. Atkins eds. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, Kramer, 1996, Aanu. Rev. Biochem., 65:367-404; Staley and Guthric, 1998, Cell 92:315- 326).

COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:44 FROM- T-966 P0311/099 F-541 4 00 r Pre-mRNA splicing proceeds by a two-step mechanism. In the first 00 step, the 5' splice site is cleaved, resulting in a "free" 5' exon and a lariat intermediate (Mooe, M.J. and P.A. Sharp, 1993, Nature 365:364-368). In the second step, the exon is ligated to the 3' exon with release of the intron as the lariat product. These steps are catalyzed in a complex of small nuclear ribonucleoproteins and proteins Scalled the spliceosome. The splicing reaction sites are defined by consensus 1 sequences around the 5' and 3' splice sites. The 5' splice site consensus sequence is c AG/GURAOU (where A-adenosine, U uracil, G guaine, C= cytosine, R purine and the splice site). The 3' splice region consists of three separate sequence elements: the branch point or branch site, a polypyrimidine tract and the 3' splice consensus sequence (YAG). These elements loosely define a 3' splice region, which may encompass 100 nucleotides of the intron upstream of the 3' splice site. The branch point consensus sequence in mammals is YNYURAC (where N iny nuoleotide, Y= pyrimidine). The underlined A is the site of branch formation (the BPA branch point adenosine). The 3' splice consensus sequence is YAG/G.

Between the branch point and the splice site there is usually found a polypyrinidine tract, which is important in mammalian systems for efficient branch point utilization and 3' splice site recognition (Roscigno, F. et at, 1993, J. BioL Chem, 268:11222- 11229). The first YAG trinucleotide downstream from the branch point and polypyrimidine tract is the most commonly used 3' splice site (Smith, C.W. et al., 1989, Nature 342:243-247).

In most cases, the splicing reaction occurs within the same pre-mRNA molecule, which is termed cis-splicing. Splicing between two independently transcribed pre-mRNAs is termed trans-splicing Trans-splicing was first discovered in trypanosomes (Sutton Boothroyd, 1986, Cell 47:527; Murphy ea al., 1986, Cell 47:517) and subsequently in nematodes (Krause Hirsh, 1987, Cell 49:753); flatworms (Rajkovic et al., 1990, Proc. Natl. Acad. ScL USA, 87:8879; Davis at a., 1995, J. Biol. Chem. 270:21813) and in plant mitochondria (Malek et al., 1997, Proc.

Nat'l. Acad. Sci. USA 94:553). In the parasite Trypanosoma brucei, allmRNAs acquire a splice leader (SL) RNA at their 5' termini by trans-splicing. A 5' leader sequence is also trans-spliced onto some genes in Caenorhabditis elegans. This COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:44 FROM- T-966 P012/099 F-541 00 0 0 mechanism is appropriate for adding a single common sequence to many different 00 transcripts.

00 The mechanism of trans-splicing, which is nearly identical to that of conventional cis-splicing, proceeds via two phosphoryl transfer reactions. The first causes the formation of a phosphodiester bond producing a Y' shaped branched intermediate, equivalent to the lariat intermediate in cis-splicing. The second -reaction, exon ligation, proceeds as in conventional cis-splicing. In addition, sequences at the 3' splice site and some of the snRNPs which catalyze the trans- O splicing reaction, closely resemble their counterparts involved in cis-splicing.

C' 10 Trans-splicing may also refer to a different process, where an intron of one pre-mRNA interacts with an intron of a second pre-mRNA, enhancing the recombination of splice sites between two conventional pre-mRNAs. This type of trans-splicing was postulated to account for transcripts encoding a human imnunoglobulin variable region sequence linked to the endogenous constant region in a transgenic mouse (Shimizu et at, 1989, Proc. Nat'l. Acad. Sci. USA 86:8020), In addition, trans-splicing of c-myb pre-RNA has been demonstrated (Vellard, M. et al.

Proc. Nat'. Acad. Sci., 1992 89:2511-2515) and more recently, RNA transcripts from cloned SV40 trans-spliced to each other were detected in cultured cells and nuclear extracts (Eul et at, 1995, EMBO. J. 14:3226). However, naturally occurring transsplicing of mammalian pre-mRNAs is thought to be an exceedingly rare event In vitro trans-splicing has been used as a model system to examine the mechanism of splicing by several groups (Konarska Sharp, 1985, Cell 46:165-171 Solnick, 1985, Cell 42:157; Chiara Reed, 1995, Nature 375:510; Pasman and Garcia-Blanco, 1996, Nucleic Acids Res. 24:1638). Reasonably efficient transsplicing (30% of cis-spliced analog) was achieved between RNAs capable of base pairing to each other, splicing of RNAs not tethered by base pairing was further diminished by a factor of 10. Other in vitro trans-splicing reactions not requiring obvious RNA-RNA interactions among the substrates were observed by Chiara Reed (1995, Nature 375:510), Bruzik J.P. Maniatis, T. (1992, Nature 360:692) and Bruzik J.P. and Maniatis, (1995, Proc. Natl. Acad. Sci. USA 92:7056-7059).

COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:44 FROM- T-966 P013/099 F-541 6 00 O0 0 These reactions occur at relatively low frequencies and require specialized elements, 00 such as a downstream 5' splice site or exonic splicing enhancers.

In addition to splicing mechanisms involving the binding of multiple proteins to the precursor mRNA which then act to correctly cut and join RNA, a third mechanism involves cutting and joining ofthe RNA by the inton itself by what are Stermed catalytic RNAmolecules or ribozymes. The cleavage activity of ribozymes 4 has been targeted to specific RNAs by engineering a discrete "hybridization" region Sinto the ribozyme. Upon hybridization to the target RNA, the catalytic region of the Sribozyme cleaves the target. It has been suggested that such ribozyme activity would be useful for the inactivation or cleavage of target RNA in vivo, such as for the treatment of human diseases characterized by production of foreign of aberrant RNA.

The use of antisense RNA has also been proposed as an alternative mechanism for targeting and destruction of specific RNAs. In such instances small RNA molecules are designed to hybridize to the target RNA and by binding to the target RNA prevent translation of the target RNA or cause destruction of the RNA through activation of nucleases.

Until recently, the practical application of targeted trans-splicing to modify specific target genes has been limited to group I ribozyme-based mechanisms.

Using the Tetrahymena group I ribozyme, targeted trans-splicing was demonstrated in E. coi. coli (Sullenger B.A. and Cech. 1994, Nature 341:619-622), inmouse fibroblasts (Jones, J.T. et al., 1996, Nature Medicine 2:643-648), human fibroblasts (Phylacton, L.A. et al. Nature Genetics 18:378-381) and human erythroid precursors (Lan et aL, 1998, Science 280:1593-1596). While many applications of targeted RNA trans-splicing driven by modified group I ribozymes have been explored, tageted trans-splicing mediated by native mammalian splicing machinery, spliceosomes, has not been previously reported.

3. SUMMAY O THE INVENTON The present invention relates to compositions and methods for generating novel nucleic acid molecules through spliceosooe-mediated targeted trans-splicing. The compositions of the invention include pre-trans-splicing COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:44 FROM- T-966 P014/099 F-41 7 00 0 0 molecules (hereinafter referred to as "PTMs") designed to interact with a natural target pre-mRNA molecule (hereinafter referred to as "pre-mRNA") and mediate a 00 spliceosomal tras-splicing reaction resulting in the generation of a novel chimeric RNA molecule (hereinafter referred to as "chimeric RNA"), The methods of the invention encompass contacting the PTMs of the invention with a natural target premRNA under conditions in which a portion of the PTM is spliced to the natural premnRNA to form a novel chimeric RNA. The PTMs of the invention are genetically engineered so that the novel chimeric RNA resulting from the trans-splicing reaction O may itself perform a function such as inhibiting the triaslation of RNA, or C 10 alternatively, the chimeric RNA may encode a protein that complements a defective or inactive protein in the cell, or encodes a toxin which kills the specific cells.

Generally, the target pre-mRNA is chosen because it is expressed within a specific cell type thereby providing a means for targeting expression of the novel chimeric RNA to a selected cell type. The target cells may include, but are not limited to those infected with viral or other infectious agents, benign or malignant neoplasms, or components of the immune system which are involved in autoimmune disease or tissue rejection. The PTMs of the invention may also be used to correct genetic mutations found to be associated with genetic diseases. In particular, double-transsplicing reactions can be used to replace internal exons. The PTMs of the invention can also be genetically engineered to tag exon sequences in a mRNA molecule as a method for identifying intron/exon boundaries in target pre-mRNA. The invention further relates to the use of PTM molecules that are genetically engineered to encode a peptide affinity purification tag for use in the purification and identification of proteins expressed in a specific cell type. The methods and compositions of the invention can be used in gene regulation, gene repair and targeted cell death. Such methods and compositions can be used for the treatment of various diseases including, but not limited to, genetic, infectious or autoimmune diseases and proliferative disorders such as cancer and to regulate gene expression in plants.

COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:45 FROM- T-966 P015/099 F-541 8 00 0 0 ,0 4. BRIEF DESCRIPTION OF THE DRAWINGS 00 Figure IA. Model of Pre-Tran-splicing RNA.

Figure IB. Model PTM constructs and targeted trans-splicing strategy. Schematic representation of the first generation PTMs (PTM+Sp and PTM- 5 Sp). BD, binding domain; NBD, non-binding domain; BP, branch point; PPT, Spyrimidine tract; ss, splice site and DT-A, diphtheria toxin subunit A. Unique restriction sites within the PTMS are indicated by single letters: E; EcoRI; X, Xhol; SI, Kp*; P. Pstl; A, Accl; B, BamHI and H; Hindll.

O Figure 1C. Schematic drawing showing the binding of PTM+Sp via conventional Watson Crick base pairing to the pHCG6 target pre-mRNA and the proposed cas- and trans-splicing mechanism.

Figure 2A. In vitro trans-splicing efficiency of various PTM constructs into HCG6 target. A targeted binding domain and active splice sites correlate with PTM trans-splicing activity. Full length targeted (pcPTM+Sp), nontargeted (PTM-Sp) and the splice mutants and PTM RNAs were added to splicing reactions containing pHCG6 target pre-mRNA. The products were R.T-PCR amplified using primers pHCG-F (specific for target pHCG6 exon 1) and DT-5R (complementary to DT-A) and analyzed by electrophoresis in a agarose gel.

Figure 2B. In vitro trans-splicing efficiency of various PTM constructs. Full length PTM with a spacer between the binding domain and splice site (PTM+Sp), PTM without the space region (PTM+) and short PTMs that contain a target binding domain (short PTM+) or a non-target binding region (PTM-) were added to splicing reactions containing PHCG target pre-mRNA. The products were RT-PCR amplified using primers pHCG-F and DT-3. For reactions containing the short PTMs, the reverse PCR primer was DT-4, since the binding site for DT-3 was removed from the PTM.

Figure 3. Nucleotide sequence demonstrating the in vitro trans-spliced product between a PTM and target pre-mRNA. The 466 bp trans-spliced RT-PCR product from Figure 2 (lane 2) was re-amplified using a 5' biotin labeled forward primer (fHCG-F) and a nested unlabeled reverse primer (DT-3R). Single stranded COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:45 FROM- T-966 P016/099 F-541 9 00 0 0 DNA was purified and sequenced directly using toxin specific DT-3R primer. The arrow indicates the splice junction between the last nuccotide of target pHCG6 exon 00 I and the first nucleotide encoding DT-A.

Figure 4A. Schematic diagram of the "safety" PTM and variations, demonstrating the PTM intramolecular base-paired stem, intended to mask the BP and PPT from splicing factors. Underlined sequences represent the pHCG6 intron I complementary target-binding domain, sequence in italics indicate target mismatches c, that are homologous to the BP.

O Figure 4B. Schematic of a safety PTM in open configuration upon C 10 binding to the target.

Figure 4C. In vitro trans-splicing reactions were carried out by incubating either safety PTM or safety PTM variants with the pHCG6 target.

Splicing reactions were amplified by RT-PCR using pHCG-F and DT-3R primers; products were analyzed in a 2.0% agarose geL Figure 5. Specificity of targeted trans-splicing is enhanced by the inclusion of a safety into the PTM. pHCG6 pre-mRNA (250 ng) and p-globin premRNA (250 ng) were annealed together with either PTM+SP (safety) or pcPTM+Sp (linear) RNA (500 ng). In vitro rans-splicing reactions and RT-PCR analysis were performed as described under experimental procedures and the products were separated on a 2,0% agarose gel. Primers used for RT-PCR are as indicated.

Figure 6. In the presence of increasing PTM concentration, cissplicing is inhibited and replaced by trans-splicing. In vitro splicing reactions were performed in the presence of a constant amount of pHCG6 target pre-mRNA (100 ng) with increasing concentrations of PTM (pcPTM+Sp) RNA (52-300 ng). RT-PCR for cis-spliced and un-spliced products utilized primers pHCG-F (exon 1 specific) and PHCG-R2 (exon 2 specific Panel primers pHCG-F and DT-3R were used to RT- PCR trans-spliced products (Panel Reaction products were analyzed on 1.5% and agarose gels, respectively. In panel A, lane 9 represents the 60 min time point in the presence of 300 ng of PTM, which is equivalent to lane 10 in panel B, Figure 7A. PTMs are capable of trans-splicing in cultured human cancer cells. Total RNA was isolated from each of 4 expanded neomycin resistant COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:45 FROM- T-966 P017/099 F-541 00 0 CdH1299 lung carcinoma colonies transfected with pcSp+CRM (expressing non-toxic 00 mutant DT-A) RT-PCR was performed using 1 pg of total RNA and 5' biotinylated pHCG-F and non-biotinylated DT-3R primers. Single stranded DNA was purified and sequenced.

Figure 7B. Nucleotide sequence (sense strand) of the trans-spliced Sproduct between endogenous PHCG6 target and CRM197 mutant toxin is shown.

Two arnows indicate the position of the splice junction.

C Figure 8A. Schematic diagram of a double splicing pre-therapeutic

O

0 mNA.

Figure SB. Selective trans-splicing of a double splicing PTM. By varying the PTM concentration the PTM can be trans-spliced into either the 5' or the 3' splice site of the target.

Figure 9. Schematic diagram of the use of PTM'molecules for exon tagging. Two examples of PTMs are shown. The PTM on the left is capable of nonspecifically trans-splicing into a target pre-mRNA 3' splice site. The other PTM on the right is designed to non-specifically trans-splice into a target pre-mRNA 5' splice site. A PTM mediated trans-splicing reaction will result in the production of a chimeric RNA comprising a specific tag to either the 5' or 3' side of an authentic exon.

Figure 10A. Schematic diagram of constructs for use in the lacZ knock-out model. The target lacZ pre-mRNA contains the 5' fragment of lacZ followed by PHCG6 intron 1 and the 3' fragment of lacZ (target The PTM molecule for use in the model systeni was created by digesting pPTM +SP with PstI and HindlII and replacing the DT-A toxin with pHCG6 exon 2 (pc3.1PTM2).

Figure 10B. Schematic diagram of restoration of P-Gal activity by Spliceosome Mediated RNA Trans-splicing. Schematic diagram of constructs for use in the lacZ knock-in model (pc.3.1 lacZ T2). The lacZ target pre-mRNA is identical to that target pre-mRNA used for the knoc'"-out experiments except that it contains two stop oodons (TAA TAA) in frame four codons after the 3' splice site. The PTM molecule for use in the model system was created by digesting pPTM +SP with Pstl and HindlI and replacing the DT-A toxin with functional 3' fragment of lacZ.

COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:45 FROM- T-966 P018/099 F-541 11 00 0' 0O ci Figure 11A. Demonstration of cis-and trans-splicing when utilizing the C4 lacZ knock-out model. The LacZ splice target 1 pre-mRNA and PTM2 were 00 co-transfected into 293T cells. Total RNA was then isolated and analyzed by PCR for cis-spliced and trans-spliced products using the appropriate specific primers. The amplified PCR products were separated on a 2% agarose gel.

Figure 11B-C. Assays for p-galactosidase activity. 293 cells were O transfected with lacZ target 2 DNA alone (panel B) or lacZ target 2 DNA and PTM1 tC (panel C).

SFigure 12A. Nucleotide sequence of rrans-spliced molecule demonstrating accurate trans-splicing.

Figure 12B. Nucleotide sequences of the cis-spliced product and the trans-spliced product. The nucleotide sequences were those sequences expected for each of the different splicing reactions.

Figure 13. Gene repair model for repair of the cystic fibrosis transmembrane regulator (CFTR) gene.

Figure 14. RT-PCR demonstration of trans-splicing between an exogenously supplied CFTR mini-gene target and PTM. Plasmids were cotransfected into 293 embryonic kidney cells. The primers pairs used for RT-PCR reactions are listed above each lane. The lower band (471 bp) in each lane represents a trans-spliced product. The lower band in lane 1 (471bp) was purified from a 2% Seakem agarose gel and the DNA sequence of the band was determined.

Figure 15. DNA sequence of the trans-spliced product (lane I, lower band shown in Figure 14). The DNA sequence indicates the presence of the F508 codon (CTT), exon 9 sequence is contiguous with exon 10 sequence, and the His tag sequence.

Figure 16. Schematic representation of repair of an exogenously supplied CFTR target molecule carrying an F508 deletion in exon Figure 17. Repair of endogenous CFTR transcripts by exon replacement using a double splicing PTM. The use of a double splicing PTM permits repair of the 4508 mutation with a very short PTM molecule.

COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:45 FROM- T-966 P019/099 F-541 12 00 0 0 Figure 18. Model lacZ target consisting of lacZ 5' exon CFTR 00 mini-intron 9- CFTR exon 10 (delta 508) CFTR mini-intron 10 followed by the lacZ 3' exon. Binding domains for PTMs are bracketed.

Figure 19. Schematic representation of double-trans-splicing PTMs designed to restore p-gal function.

Figure 20. Schematic representation of a double-trans-splicing reation showing the binding of DSPTM7 with DSCFT1.6 target pre-mRNA.

Figure 21. Important structural elements of DSPTM7. The double splicing PTM has both 3' and 5' functional splice sites as well as binding domains.

Fignure 22. Schematic diagram of mutant double splicing PTMs.

Figure 23. Accuracy of double-trans-splicing reaction.

Figure 24. Doubletrans-splicing between the target pre-mENA and the DSPTM7 produces fullt-length protein. Western blot analysis of total cell lysates using polyclonalw anti-p-gaactosidase antisaeram.

Figure 25. Precise intemrnal exon substitution between the DSCFT1l 6 target pre-nRNA and DSPTM7 RNA by double-trans-splicing produces functionally active p-gal protein. Total cell extracts were prepared and assayed for 0-gal activity using an ONPG assay.

Figure 26. 3' and 5' splice sites are essential for the restoration of p-gal function by double-wns-splicing reaction.

Figure 27. Double-rans-splicing: titration of target and PTMvl.

Different concentraftiOns of the target and PTM were co-transfected and analyzed for p-gal activity restoration.

Figure 28. Constructs designed to test the specificity of double-trans-splicing reaction.

Figure 29. Specificity of a double-trans-splicing reaction.

Figure 30. Trans-splicing repair of the cystic fibrosis gene using a pTM that mediates a double-trans-splicing event.

Figure 31. DIM with a long binding domain masking two splice sites and part of exon 10 in a mini-gene target COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:45 FROM- T-966 P 0 20 /099 F-541 13 00 0 0 1 'Figure 32. Sequence of a single PCR product showing target exon 9 C correctly spliced to PTM oxon 10 (with modified codons) (upper panel), codon 508 in exon 10 of the PTM (middle panel) and PTM exon 10 correctly spliced to target exon 11 (lower panel). The sequence of a repaired target was generated by RT-PCR followed by PCR.

S. Figure 33. Trans-splicing repair of the cystic fibrosis gene using a PTM that can perform 5' exon replacement CI Figure 34. Schematic diagram of three different PTM molecules with o different binding domains.

CI 10 Figure 35. Schematic diagram ofPTM exon 10 with modified codon usage to reduce antisense effects with its own binding domain.

Figure 36. Sequence of cis- and trans-spliced products.

Figure 37. Model system for repair of messenger RNAs by transsplicing. Schematic illustration of a defective lacZCF9m splice target uied in the present study (see Materials and Methods for details). BP, branch point; PPT, polypyrimidine tracts; as, splice sites and pA, polyadenylation signal. A prototype PTM showing the key components of the trans-splicing domain, and the diagrams of various PTMs showing the binding domain length and approximate positions at which they bind to the target pre-mRNA. Unique restriction sites within the trans-splicing domain are N, Nhe I; S, Sac ll; K, Kpn I and E, EcoR V. (C) Schematic diagram showing the binding of a PTM through antisense binding and repair of defective lacZpre-mRNA throagh targeted RNA trans-splicing. Expected cis and trans-spliced products and the primer binding sites for Lac-9F, Lac-3R and are indicated.

Figure 38. Efficient repair of IacZ messenger RNA. Target specific primers, Lac-9F exon) and Lac-3R exon) were used to amplify ci-spliced products (lanes while; target and PTM specific primers, Lac-9F exon) and Lac-SR exon) were used to amplify trans-spliced products (lanes 7-15). 25-50 ng of total RNA was used to measure target cts-splicing (lanes 1-6) and 50-200 ng of total RNA was used to measure PTM induced RNA trans-splicing (lanes 7-12).

Lanes 13-15, 25-50 ng of total RNA from cells transfected with lacZCF9 a control for COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:46 FROM- T-966 P021/099 F-541 14 00 0 CtrWs-spliing. Endogenous mRNA repair by trans-splicing. Lanes 1-3, RNA from cells transfected with PTM-CF14; lanes 4-6. PTM-CF22 and lanes 7-9, PTM- 00 CF24. Lane 10, RNA from mock-transfected cells and lane 11 is a control in which reverse-transcription reaction was omitted.

Figure 39. Messenger RNA repair leads to synthesis of full-length P-galactosidase. Lane 1, lacZCF9 (positive control, 5 tg); lane 2, lacZCF9m target alone (25 ug); lane 3, PTM-CF24 alone (25 ig) and lane 4, laoZCF9m target ¢C PTM-CF24 (25 pg).

O Figure 40. Messenger RNA repair by SMaRT produces functional C 10 P-galactosidase. In situ detection of functional p-galactosidase produced by trans-splicing. 293T cells were either transfected (transient assay) with lacZCF9m target alone (panel A) or co-transfected with lacZCF9m target PTM-CF24 (panel B) expression plasmids as described above. 48-hr post-transfection, cells were rinsed with PBS and stained in situ for p-gal activity. Repair of a defective lacZ mRNA produces functional p-galactosidase. Target and PTM, extracts from cells transfected with either lacZCF9m target or PTM-CF24 plasmid alone, and the rest were from cells co-transfected with lacZCF9m target and one of the PTMs as indicated. (C) Endogenous mRNA repair by trans-splicing produces functional p-galactosidase.

Stable cells expressing an endogenous lacZCF9m pre-mRNA target was transfected with "linear" PTMs (PTM-CF14, PTM-CF22 or PTM-CF24) as described above.

Following transfection, total cell lysate was prepared and assayed for P-gal activity.

The results presented are the average of two independent transfections.

Figure 41. Messenger RNA repair is specific. Experimental strategy to measure non-specific trans-splicing between lacZHCGlm pre-mRNA and "linear" PTMs. Extended binding domains enhance the specificity oftranssplicing. Lanes 1-3, PTM-CF14; 4-6, PTM-CF22; 7-9, PTM-CF24; 10-12, PTM- CF26 and 13-15, PTM-CF27. PTMs with very long binding domains are capable of increasing specificity. Total cell extract (5 pA) was assayed in solution for p-gal activity and the specific activity was calculated. P-gal activity was normalized to mock and the results presented are the average of two independent transfeotions.

COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:46 FROM- T-966 P022/99 F-541 00 0 0 0) Control, extract from cells transfected with laoZHCGlm target alone and the rest were co-transfected with lacZHCGlm target and one of the linear PTMs.

00 Figure 42. Complete sequence of CTR PTM 30 exon replacement PTM) showing the trans-splicing domain (underlined) and the coding sequence for 5 exons 1-10 of the CFTR gene. Modified codons in exon 10 are underlined and bold.

Figure 43A. 153 base-pairPTM 24 Binding Domain.

Figure 43B. Complete sequence of CFTR PTM 24 xon replacement PTM) showing the trans-splicing domain (underlined) and the coding Ssequence for exons 10-24 of the CFTR cDNA. At the end of the coding is a histidine C' 10 tag and the translation stop codon.

Figure 44A. Detailed structure of the mouse factor VII PTM containing normal mouse sequences for exons 16-26. BGH=bovine growth hormone 3' UTR (untranslated sequence); Binding Domain125bp; base changes to eliminate cryptic sites are circled:F5, F6, F7, F8--primer sites.

Figure 44B. Schematic diagram showing the extent of the binding domain in the mouse factor VIII gene.

Figure 44C. Changes to the promoter in AAV vectors pDLZ20 and pDLZ20-M2 to eliminate cryptic donor sites in sequence upstream of the PTM binding domain Figure 44D. Factor VIII repair model. Schematic diagram of a PTM binding to the 3' splice site ofintron 15 of the mouse factor VIII gene.

Figure 45. Schematic diagram of a F8 PTM with the trans-splicing domain eliminated. This represents a control PTM to test whether repair is a result of trans-splicing.

Figure 46. Data indicating repair of factor VIII in Factor VI knock out mice. Blood was assayed for factor VIII activity using a coatest assay.

Figure 47A. Detailed structure of a mouse factor VIIIPTM containing normal sequences for exons 16-26 and a C-terminal FLAG tag. BGH=bovine growth hormone 3"UTR; Binding domain-125 bp.

Figure 47B. Detailed structure of a human or canine factor VIII PTM containing normal sequences for exons 23-26.

COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:46 FROM- T-966 P023/099 F-541 16 00

O

O

0) Figure 48. Transcription Map of HPV-16.

Figure 49. Disruption of Human Papillomavirus Type 16 Expression 00 by PTM. Schematic diagram of HPV-PTM 2 binding to the 3' splice site of the HPV type 16 target pre-mRNA.

Figure 50. E7 Targeting Strategy in which Multiple PTMs are targeted to HPV E7.

Figure 51. PTM Design indicating the binding domain, branch point C and polypyrimidine tract.

O Figure 52A. HPV-PTM 1 with 80 bp binding domain targeted to 3' as C 10 at 409.

Figure 52B. HPV-PTM 2 with 149 bp binding domain targeted to 3' ss at 409, Figure 53. Binding Domains of HPV-PTM 3 and 4.

Figure 54. Binding Domains of HPV-PTM 5 and 6. Nucleotides in bold are modified to prevent cryptic splicing of PTMs.

Figure 55. Positions of HPV-PTM targeting domains.

Figure 56. Trans-splicing Efficiency of HPV-PTMs in 293 T Cells.

293T cells were con-transfected with 2 pg ofp1059 target and 1.5 pg of PTM expression plasmids. 48 hr post-transfection, total RNA was isolated and analyzed by RT-PCR. Target specific primers, oJMD15 and JMD16 were usedto amplify cisspliced products (lanes 1-11, upper panel), while; target and PTM specific primers, and Lac-6R were used to amplify transspliced products (lanes 1-12, lower panel). Lanes 13-14 (upper panel), RNA isolated from cells that are transfected with lacZCF9 and HPV-PTM1 and 2 respectively, hence, serve as controls for evaluating the specificity ofHPV-PTMs.

Figure 57. Nucleotide sequence showing the trans-splice junctions between the HPV target pre-mRNA and the PTM. The RT-PCR product was purified and sequenced directly using primer Lac5R (binds to 3' exon of the PTM). The arrow indicate trans-splice junction between E6 ofHPV pre-mRNA target and lacZ 3' exon of the PTM..

COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:46 FROM- T-966 P024099 F-541 17 00 0 0 Figure 58. Tran-splicing in 293 cells (Co-transfections) Quantification of tran-splicing efficiency was detennined using real-time QRT-PCR.

00 SFigure 59. Trans-splicing efficiency of HPV-PTMs into an endogenous pre-mRNA target. SiHa and CaSki cells were transfected wit 1.5 Rag of either HPV-PTM1, 2 or CFTR targets PTM14 or 27 expression plasmids. 48 hr posttransplicing, total RNA was isolated and analyzed by RT-PCR. Trans-splicing Sbetween the endogenous HPV target and the PTm was detected using target and PTM c *specific primers oJMD15 and Lac-16R. The expected trans-spliced product (418 bp) O is clearly visible in cells that are transfected with HPV-PTMs (lanes 2-3 and 5-7) but 0 C' 10 not in control (lanes 1 and In addition, trans-splicing is also detected in lane 8 due to non-specific trans-splicing.

Figure 60. Accurate r-ans-splicing of HPV-PTMI in SiHa Cells.

Target pre-mRNA was endogenous mRNA. Sequence analysis of trans-spliced chimeric RNA indicates that trans-splicing is accurate.

Figure 61. Quantification oftrans-splicing efficiency in SiHa cells using real-time QRT-PCR.

Figure 62. Trans-splicing efficiency ofHPV-PTM 1, HPV-PTM 5, HPV-PTM 6 in SiHa cells. Analysis of total RNA was performed using RT-PCR.

Figure 63. Deletion ofpolypyrimidine tract abolishes trans-splicing.

Lanes I and 2 represent RNA from cells transfeoted with mutant HPV-PPT. Lanes 3 and 4 represent RNA from cells transfected with HPV-PTM5 plasmid. 269 bp product resulting from trans-splicing is detected.

Figure 64. Schematic Diagram of a PTM binding to the 5' splice site of the HPV mini-gene target and the resulting trans-spliced chimera RNA.

Figure 65. Double Trans-splicing. Schematic diagram of a double trans-splicing PTM binding to the 3' and 5' splice sites of the HPV mini-gene target.

The resultant trans-spliced mRNA is shown.

Figure 66A Trans-splicing by 3' exon replacement. Schematic diagram of a PTM binding to the 3' splice site of the HPV mini-gene target Figure 66B. Trans-splicing by 5' exonreplacement. Schematic diagram of a PTM binding to the 5' splice site of the HPV mini-gene target.

COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:46 FROM- T-966 P025/99 F-541 18 00 0 0 ci D Figure 67. Schematic of a double splicing HPV-PTM designed for 00 internal exon replacement DBTAUILD DESCRIPTION OF THE INVENTION O The present invention relates to compositions comprising pre-tras- N 5 splicing molecules (PTMs) and the use of such molecules for generating novel nucleic Sacid molecules. The PTMs of the invention comprise one or more target binding Cl domains that are designed to specifically bind to pre-mRNA, a 3' splice region that 0 includes a branch point, pyrimidine tract and a 3' splice acceptor site and/or a 5' splice Cdonor site; and one or more spacer regions that separate the RNA splice site from the target binding domain. In addition, the PTMs of the invention can be engineered to contain any nucleotide sequences such as those encoding a translatable protein product.

The methods of the invention encompass contacting the PTMs of the invention with a natural pre-mRNA under conditions in which a portion of the PTM is trans-spliced to a portion of the natural pre-mRNA to form a novel chimeric RNA.

The target pre-muRNA is chosen as a target due to its expression within a specific cell type thus providing a mechanism for targeting expression of a novel RNA to a selected cell type. The resulting chimeric RNA may provide a desired function, or may produce a gene product in the specific cell type. The specific cells may include, but are not limited to those infected with viral or other infectious agents, benign or malignant neoplasms, or components of the immune system which are involved in autoimmune disease or tissue rejection. Specificity is achieved by modification of the binding domain of the PTM to bind to the target endogenous pre-mRNA. The gene products encoded by the chimeric RNA can be any gene, including genes having clinical usefulness, for example, therapeutic or marker genes, and genes encoding toxins.

5.1. STRUCTURE OP THE TRE-TPRANS-SPICN

MOLECULES

The present invention provides compositions for use in generating novel chimeric nucleic acid molecules through targeted trans-splicing. The PTMs of the invention comprise one or more target binding domains that targets binding of COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:46 FROM- T1-966 P026/099 F-541 19 00 C) the PTM to a pre-mRNA (ii) a 3' splice region that includes a branch point, pyrimidine tract and a 3' splice acceptor site and/or 5' splice donor site; and (iii) one or more spacer regions to separate the RNA splice site from the target binding domain. Additionally, the PTMs can be engineered to contain any nucleotide 5 sequence encoding a translatable protein product. In yet another embodiment of the ll~n invention, the PTMs can be engineered to contain nucleotide sequences that inhibit Othe translation of the chimic RNA molecule. For example, the nucleotide sequences ^C may contain translational stop codons or nucleotide sequences that form secondary 1 structures and thereby inhibit translation. Alternatively, the chimeric RNA may C- 10 function as anantisense molecule thereby inhibiting translation of the RNA to which it binds.

The target binding domain of the PTM may contain multiple binding domains which are complementary to and in anti-sense orientation to the targeted region of the selected pre-mRNA. As used herein, a target binding domain is defined as any sequence that confers specificity of binding and anchors the pre-mRNA closely in space so that the spliceosome processing machinery of the nucleus can trans-splice a portion of the PTM to a portion of the pre-mRNA. The target binding domains may comprise up to several thousand nucleotides. In preferred embodiments of the invention the binding domains may comprise at least 10 to 30 and up to several hundred nucleotides. As demonstrated herein, the specificity of the PTM can be increased significantly by increasing the length of the target binding domain. For example, the target binding domain may comprise several hundred nucleotides or more. In addition, although the target binding domain may be "linear" it is understood that the RNA may fold to form secondary structures that may stabilize the complex thereby increasing the efficiency of splicing. A second target binding region may be placed at the 3' end of the molecule and can be incorporated into the PTM of the invention. Absolute complementarity, although preferred, is not required.

A sequence "complementary" to a portion of an RNA, as referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the RNA, formnning a stable duplex. The ability to hybridize will depend on both the degree of complementarity and the length of the nucleic acid (See, for example, Sambrook et COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:47 FROM- T-966 P027/099 F-541 00 0 0 Sal., 1989, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor 00 Laboratory Press, Cold Spring Harbor, New York). Generally, the longer the hybridizing nucleic acid, the more base mismatches with an RNA it may contain and still form a stable duplex. One skilled in the art can ascertain a tolerable degree of mismatch or length of duplex by use of standard procedures to determine the stability of the hybridized complex.

Where the PTMs are designed for use in intron-exon tagging or for Speptide affinity tagging, a library of PTMs is genetically engineered to contain Srandom nucleotide sequences in the target binding domain. Alternatively, for intronexon tagging the PTMe may be genetically engineered so as to lack target binding domains. The goal of generating such a library of PTM molecules is that the library will contain a population of PTM molecules capable of binding to each RNA molecule expressed in the cell. A recombinant expression vector can be genetically engineered to contain a coding region for a PTM including a restriction endonuclease site that can be used for insertion of random DNA fragments into the PTM to form random target binding domains. The random nucleotide sequences to be included in the PTM as target binding domains can be generated using a variety of different methods well known to those of skill in the art, including but not limited to, partial digestion of DNA with restriction enzymes or mechanical shearing of DNA to generate random fragments of DNA. Random binding domain regions may also be generated by degenerate oligonucleotide synthesis. The degenerate oligonucleotides can be engineered to have restriction endonuclease recognition sites on each end to facilitate cloning into a PTM molecule for production of a library of PTM molecules having degenerate binding domains.

Binding may also be achieved through other mechanisms, for example, through triple helix formation or protein/nucleic acid interactions such as those in which the PTM is engineered to recognize a specific RNA binding protein, a protein bound to a specific target pre-mRNA. Alternatively, the PTMs of the invention may be designed to recognize secondary structures, such as for example, hairpin structures resulting from intramolecular base pairing between nucleotides within an RNA molecule.

COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:47 FROM- T-966 P028/099 F-541 21 00 O0

O

The PTM molecule also contains a 3' splice region that includes a branch point, pyrimidino tract and a 3' splice acceptor AG site and/or a 5' splice donor 00 site. Consensus sequences for the 5' splice donor site and the 3' splice region used in RNA splicing are well known in the art (See, Moore, et al., 1993, The RNA World, Cold Spring Harbor Laboratory Press, p. 303-358). In addition, modified consensus sequences that maintain the ability to function as 5' donor splice sites and 3' splice Ssregions may be used in the practice of the invention. Briefly, the 5' splice site Sconsensus sequence is AG/GURAGU (where A-adenosine, U-aracil, G=guanine, O C=cytosine, R=purine and /-the splice site). The 3' splice site consists of three Ci 10 separate sequence elements: the branch point or branch site, a polypyrimidine tract and the 3' consensus sequence (YAG). The branch point consensus sequence in mammals is YNYURAC (Y=pyrimidine). The underlined A is the site of branch formation. A polypyrimidine tract is located between the branch point and the splice site acceptor and is important for different branch point utilization and 3' splice site recognition.

Further, PTMs comprising a 3' acceptor site (AG) may be genetically engineered. Such PTMs may further comprise a pyrimidine tract and/or branch point sequence.

Recently, pre-messenger RNA introns beginning with the dinucleotide AU and ending with the dinucleotide AC have been identified and referred to as U 12 introns. U12 intron sequences as well as any sequences that function as splice acceptor/donor sequences may also be used in PTMs.

A spacer region to separate the RNA splice site from the target binding domain is also included in the PTM. The spacer region can have features such as stop codons which would block any translation of an unspliced PTM and/or sequences that enhance trans-splicing to the target pre-mRNA. In a preferred embodiment of the invention, a "safety" is also incorporated into the spacer, binding domain, or elsewhere in the PTM to prevent non-specific trans-splicing. This is a region of the PTM that covers elements of the 3' and/or 5' splice site of the PTM by relatively weak complementarity, preventing nonspecific trans-splicing. The PTM is designed in such a way that upon hybridization COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:47 FROM- T-966 P029/099 F-541 22 00 0 0 1) of the binding/targeting portion(s) of the PTM, the 3' and/or 5'splice site is uncovered 00 and becomes fully active.

The "safety" consists of one or more complementary stretches of cisequence (or could be a second, separate, strand of nucleic acid) which weakly binds to one or both sides of the PTM branch point, pyrimidine tract, 3' splice site and/or O splice site (splicing elements), or could bind to parts of the splicing elements Sthemselves. This "safety" binding prevents the splicing elements from being active MC block U2 snRNP or other splicing factors from attaching to the PTM splice site Srecognition elements). The binding of the "safety" may be disrupted by the binding of the target binding region of the PTM to the target pre-mRNA, thus exposing and activating the PTM splicing elements (making them available to trans-splice into the target pre-mRNA).

A nucleotide sequence encoding a translatable protein capable of producing an effect, such as cell death, or alternatively, one that restores a missing function or acts as a marker, is included in the PTM of the invention. For example, the nucleotide sequence can include those sequences encoding gene products missing of altered in known genetic diseases. Alternatively, the nucleotide sequences can encode marker proteins or peptides which may be used to identify or image cells. In yet another embodiment of the invention nuleotide sequences encoding affinity tags such as, HIS tags (6 consecutive histidine residues) (Janlmecht,.et aL, 1991, Proc.

Natl. Acad. Sci. USA 88:8972-8976), the C-temmius of glutathione-S-transferase (GST) (Smith and Johnson, 1986, Proc. Natl. Acad. Sci. USA 83:8703--8707) (Pharmacia) or FLAG (Asp-Tyr-Lys-Asp-Asp-Asp-Lys) (Eastman Kodak/BI, Rochester, NY) can be included in PTM molecules for use in affinity purification.

The use of PTMs containing such nucleotide sequences results in the production of a chimeric RNA encoding a fusion protein containing peptide sequences normally expressed in a cell linked to the peptide affinity tag. The affinity tag provides a method for the rapid purification and identification of peptide sequences expressed in the cell. In a preferred embodiment the nucleotide sequences may encode toxins or other proteins which provide some function which enhances the susceptibility of the .cells to subsequent treatments, such as radiation or chemotherapy.

COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:47 FROM- T-966 P030/099 F-541 23 00 0 0 In a highly preferred embodiment of the invention a PTM molecule is Sdesigned to contain nucleotide sequences encoding the Diphtheria toxin subunit A 00 (Greenfield, et al., 1983, Proc. Natl. Acad. Sci. USA 80: 6853-6857). Diphtheria toxin subunit A contains enzymatic toxin activity and will function if expressed or delivered into human cells resulting in cell death. Furthermore, various other known peptide toxins may be used in the present invention, including but not limited to, ricin, NO Pseudomonus toxin, Shiga toxin and exotoxin A.

Ci Additional features can be added to the PTM molecule either after, or o before, the nucleotide sequence encoding a translatable protein, such as "i 10 polyadenylation signals or 5' splice sequences to enhance splicing, additional binding regions, "safety"-self complementary regions, additional splice sites, or protective groups to modulate the stability of the molecule and prevent degradation.

Additional features that may be incorporated into the PTMs of the invention include stop codons or other elements in the region between the binding domain and the splice site to prevent unspliced pre-mRNA expression. In another embodiment of the invention, PTMs can be generated with a second anti-sense binding domain downstream from the nucleotide sequences encoding a translatable protein to promote binding to the 3' target intron or exon and to block the fixed authentic cts-5' splice site (US and/or U1 binding sites).

PTMs may also be generated that require a double-rans-splicing reaction for generation of a chimeric trans-spliced product. Such PTMs could be used to replace an internal exon which could be used for RNA repair. PTMs designed to promote two trans-splicing reactions are engineered as described above, however, they contain both 5' donor sites and 3' splice acceptor sites. In addition, the PTMs may comprise two or more binding domains and splicer regions. The splicer regions may be place between the multiple binding domains and splice sites or alternatively between the multiple binding domains.

Further elements such as a 3' hairpin structure, circularized RNA, nucleotide base modification, or a synthetic analog can be incorporated into PTMs to promote or facilitate nuclear localization and spliceosomal incorporation, and intracellular stability.

COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:47 FROM- T-966 P031/099 F-541 24 00 0 0 SAdditionally, when engineering PTMs for use in plant cells it may not be necessary to include conserved branch point sequences or polypyrimidino tracts as 00 these sequences may not be essential for intron processing in plants. However, a 3' splice acceptor site and/or 5' splice donor site, such as those required for splicing in vertebrates and yeast, will be included. Further, the efficiency of splicing in plants may be increased by also including UA-rich intronic sequences. The skilled artisan will recognize that any sequences that are capable of mediating a trans-splicing c reaction in plants may be used.

O The PTMs of the invention can be used in methods designed to 10 produce a novel chimeric RNA in a target cell. The methods of the present invention comprise delivering to the target cell a PTM which may be in any form used by one skilled in the art, for example, an RNA molecule, or a DNA vector which is transcribed into a RNA molecule, wherein said PTM binds to a pre-mRNA and mediates a trans-splicing reaction resulting in formation of a chimeric RNA comprising a portion of the PTM molecule spliced to a portion of the pre-mRNA.

5.2. SYNTHESIS OF THE TRANS-SPLICING MOLECULES The nucleic acid molecules of the invention can be RNA or DNA or derivatives or modified versions thereof, single-stranded or double-stranded. By nucleic acid is meant a PTM molecule or a nucleic acid molecule encoding a PTM molecule, whether composed ofdeoxyribonucleotides or ribonucleosides, and whether composed ofphosphodiester linkages or modified linkages. The term nucleic acid also specifically includes nucleic acids composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine and uracil).

The RNA and DNA molecules of the invention can be prepared by any method known in the art for the synthesis of DNA and RNA molecules. For example, the nucleic acids may be chemically synthesized using commercially available reagents and synthesizers by methods that are well known in the art (see, Gait, 1985, Oligonucleotide Synthesis: A Practical Approach, IRL Press, Oxford, England).

Alternatively, RNA molecules can be generated by in vitro and in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:48 FROM- T-966 P032/09 F-41 00 0 0 0) incorporated into a wide variety of vectors which incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. RNAs maybe produced in high yield via in vitro transcription using plasmids such as (Promega Corporation, Madison, WI). In addition, RNA amplification methods such 5 as Q-P amplification can be utilized to produce RNAs.

tn The nucleic acid molecules can be modified at the base moiety, sugar Smoiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, transport into the cell, etc. For example, modification ofa PTM to O reduce the overall charge can enhance the cellular uptake of the molecule. In addition Ci 10 modifications can be made to reduce susceptibility to nuclease degradation. The nucleic acid molecules may include other appended groups such as peptides for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, Letsinger et 1989, Proc. Natl. Acad. Sci. U.S.A. 86:6553- 6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84:648-652; PCT Publication No.

W088/09810, published December 15, 1988) or the blood-brain barrier (see, PCT Publication No. W089/10134, published April 25, 1988), hybridization-triggered cleavage agents. (See, Krol et al, 1988, BioTechniques 6:958-976) or intercalating agents. (See, Zon, 1988, Pharm. Res. 5:539-549). To this end, the nucleic acid molecules may be conjugated to another molecule, a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc. Various other well-known modifications to the nucleic acid molecules can be introduced as a means of increasing intracellular stability and halflife. Possible modifications include, but are not limited to, the addition of flanking sequences ofribo- or deoxy- nucleotides to the 5' and/or 3' ends of the molecule. In some circumstances where increased stability is desired, nucleic acids having modified internuclooside linkages such as 2'-0-nmthylation may be preferred. Nucleic acids containing modified internucleoside linkages may be synthesized using reagents and methods that are well known in the art (see, Uhlmann et al., 1990, Chem. Rev.

90:543-584; Schneider et al., 1990, Tetrahedron Lett. 31:335 and references sited therein).

COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:48 FROM- T-966 P033/099 F-541 26 00 0 0 The nucleic acids may be purified by any suitable means, as are well 00 known in the art For example, the nucleic acids canbe purified by reverse phase chromatography or gel electrophoresis. Of course,'the skilled artisan will recognize that the method of purification will depend in part on the size of the nucleic acid to be purified.

In instances where a nucleic acid molecule encoding a PTM is utilized, cloning techniques known in the art maybe used for cloning of the nucleic c, acid molecule into an expression vector. Methods commonly known in the art of O recombinant DNA technology which can be used are described in Ausubel et al.

C 10 1993, Current Protocols in Molecular Biology, John Wiley Sons, NY; and Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press,

NY.

The DNA encoding the PTM of interest may be recombinantly engineered into a variety of host vector systems that also provide for replication of t:.

DNA in large scale and contain the necessary elements for directing the transcription of the PTM. The use of such a construct to transfect target cells in the patient will result in the transcription of sufficient amounts of PTMs that will form complementary base pairs with the endogenously expressed pre-mRNA targets and thereby facilitate a rrans-splicing reaction between the complexed nucleic acid molecles. For example, a vector can be introduced in vivo such that it is taken up by a cell and directs the transcription of the PTM molecule. Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired RNA. Such vectors can be constructed by recombinant DNA technology methods standard in the art.

Vectors encoding the PTM of interest can be plasmid, viral, or others known in the art, used for replication and expression in mammalian cells. Expression of the sequence encoding the PTM can be regulated by any promoter known in the art to act in mammalian, preferably human cells. Such promoters can be inducible or constitutive. Such promoters include but are not limited to: the SV40 early promoter region (Benoist, C. and Chambon, P. 1981, Nature 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et a., COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:48 FROM- T-966 P034/099 F-541 27 00 0 0 1980, Cell 22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:14411445), the regulatory sequences of the Smetallothionein gene (Brinster t al., 1982, Nature 296:39-42), the viral CMV promoter, the human chorionic gonadotropin-P promoter (Hollenberg et al., 1994, Mol. Cell. Endocrinology 106:111-119), etc. Any type ofplasmid, cosmid, YAC or \viral vector can be used to prepare the recombinant DNA construct which can be S rintroduced directly into the tissue site. Alternatively, viral vectdrs can be used which selectively infect the desired target cell.

O For use of PTMs encoding peptide affinity purification tags, it is C 10 desirable to insert nucleotide sequences containing random target binding sites into the PTMs and clone them into a selectable mammalian expression vector system. A number of selection systems can be used, including but not limited to selection for expression of the herpes simplex virus thymidine kinase, bypoxanthine-guanine phosphoribosyltransterase and adenine phosphoribosyl transferase protein in tk-, hgprt- or aprt- deficient cells, respectively. Also, anti-metabolic resistance can be used as the basis of selection for dihydrofolate tranferase (dhfr), which confers resistance to methotrexate; xanthine-guanine phosphoribosyl transferase (gpt), which confers resistance to mycophenolic acid; neomycin (neo), which confers resistance to aminoglycoside G-418; and hygromycin B phosphotransferase (hygro) which confers resistance to hygromycin. In a preferred embodiment of the invention, the cell culture is transformed at a low ratio of vector to cell such that there will be only a single vector, or a limited number of vectors, present in any one cell. Vectors for use in the practice of the invention include any eukaryotic expression vectors, including but not limited to viral expression vectors such as those derived from the class ofretroviruses or adeno-associated viruses.

5.3. USES AND A.DMINISTRATION OF TRANS-SPLICING MOLECULES 5.3.1. USE OF PTM MOLECULES FOR GENE REGULATION, GENE REPAIR AND TARGETED CELL DEATH The compositions and methods of the present invention will have a variety of different applications including gene regulation, gene repair and targeted COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:48 FROM- T-966 P035/099 F-541 28 00 0 0 cell death. For example, trans-splicing can be used to introduce a protein with toxic Ll properties into a cell. In addition, PTMs can be engineered to bind to viral mRNA 00 and destroy the function of the viral mRNA, or alternatively, to destroy any cell expressing the viral mRNA. In yet another embodiment of the invention, PTMs can be engineered to place a stop codon in a deleterious nRNA transcript thereby decreasing the expression of that transcript.

In an embodiment of the invention PTM molecules were designed to bind to papilloma virus RNA and inhibit the function of the viral RNA. Specifically o anti-HPV PTMs were designed to specifically target HPV pre-mRNAs and result in C 10 the expression of a disruptive or toxic protein only in the HPV-infected cancer cells.

Thus, the invention provides PTM molecules designed to inhibit the function of papilloma virus RNA. Such papilloma viruses, include but are not limited to mammalian papillomaviruses including human papillomaviruses.

The papilloma viruses are a group of small DNA viruses which induce papillomas (warts) in a variety of vertebrates, including human. In addition, human papilloma virus is one of the most common causes of sexually transmitted diseases in the country and the vast majority of cervical cancers are associated with oncogenic human papillomavinuses and express viral mRNAs encoding the E6 and E7 oncoproteins. Thus, the PTM molecules of the invention may be used to inhibit the proliferation of papillomaviruses within an infected host.

Targeted trans-splicing, including double-trans-splicing reactions, 3' exon replacement and/or 5' exon replacement can be used to repair or correct transcripts that are either truncated or contain point mutations. The PTMs of the invention are designed to cleave a targeted transcript upstream or downstream of a specific mutation or upstream of a premature 3' and correct the mutant transcript via a trans-splicing reaction which replaces the portion of the transcript containing the mutation with a functional sequence.

In addition, double trans-spliing reactions may be used for the selective expression of a toxin in tumor cells. For example, PTMs can be designed to replace the second exon of the human P-chronic gonadotropin-6 (phCG6) gene transcripts and to deliver an exon encoding the subunit A of diptheria toxin (DT-A).

COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:49 FROM- T-966 P036/099 F-541 29 00 0 0 0) Expression of DT-A in the absence of subunit B should lead to toxicity only in the cells expressing the gene. PhCG6 is a prototypical target for genetic modification by trans-splicing. The sequence and the structure of the phCG6 gene are completely known and the pattern of splicing has been determined. The phCG6 gene is highly expressed in many types of solid tumors, including many non-germ line tumors, but O the phCG6 gene is silent in the majority cells in a normal adult. Therefore, the phCG6

O

Spre-mRNA represents a desirable target for a trans-splicing reaction designed to C produce tumor-specific toxicity.

o The first exon of pJhCG6 pre-mRNA is ideal in that it encodes only Cl 10 five amino acids, including the initiator AUG, which should result in minimal interference with the proper folding of the DT-A toxin while providing the required signals for effective translation of the trans-spliced mRNA. The DT-A exon, which is designed to include a stop codon to prevent chimeric protein formation, will be engineered to trans-splice into the last exon of the phCG6 gene. The last exon of the phCG6 gone provides the construct with the appropriate signals to polyadenylate the mRNA and ensure translation.

Cystic fibrosis (CF) is one of the most common fatal genetic disease in humans. Based on both genetic and molecular analyses, the gene associated with cystic fibrosis has been isolated and its protein product deduced (Kerem, B.S. et al., 1989, Science 245:1073-1080; Riordan et al., 1989, Science 245:1066- 1073;Ronmmans, et al., 1989, Science 245:1059-1065). The protein product of the CF associated gene is called the cystic fibrosis transmembrane conductance regulator (CFTR). In a specific embodiment of the invention, a trans-splicing reaction will be used to correct a genetic defect in the DNA sequence encoding the cystic fibrosis transmenbrane regulator (CFTR) whereby the DNA sequence encoding the cystic fibrosis trans-membrane regulator protein is expressed and a functional chloride ion channel is produced in the airway epithelial cells of a patient.

Population studies have indicated that the most common cystic fibrosis mutation is a deletion of the three nucleotides in exon 10 that encode phenylalanine at position 508 of the CFTR amino acid sequence. As indicated in Figure 15, a transsplicing reaction was capable of correcting the deletion at position 508 in the CFTR COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:49 FROM- T-966 P037/099 F-541 00 0 0 ci 0) amino acid sequence. The PTM used for correction of the genetic defect contained a CFTR BD intron 9 sequence, a spacer sequence, a branch point, a polypyrimidine 00 tract, a 3' splice site and a wild type CFTR BD exon 10 sequence (Figure 13). The successful correction of the mutated DNA encoding CFTR utilizing a trns-splicing reaction supports the general application of PTMs for correction of genetic defects.

HemophiliaA is an X-linked bleeding disorder characterized by a deficiency in the activity of factor VIII, a n important component of the coagulation cascade. The incidence of hemophilia A is approximately 1 in 5,000 to 10,000 males.

O Affected individuals suffer joint and muscle hemorrhage, easy bruising, and Ci 10 prolonged bleeding from wounds. Hemophilia A arises from a variety of mutations within the factor VIII gene. The gene comprises 26 exons and spans 186 kb. About percent of those patients with hemophilia A in whom mutations have been characterized, have point mutations in the gene. In a specific embodiment of the invention, a trans-splicing reaction will be used to correct a genetic defect in the DNA sequence encoding factor VIII whereby the DNA sequence encoding the factor VII protein is expressed and a functional clotting factor is produced in the plasma of a patient. The PTMs of the invention can be genetically engineered to repair any exon of interest, or combination of exons for the purpose.of correcting a defect in the coding region of the factor VII gene.

Genetic studies have indicated that the most common factor VIII mutation(s) are be generated. As indicated in Figure 46, a trans-splicing reaction was capable of correcting the mutation in the factor VIII amino acid sequence. The mutation was created by an insertion of the neomycin gene into exon 16 and intron 16 of the mouse gene, interrupting the open reading frame of exon 16 and eliminating intron 16's 3' splice donor site. The PTM used for correction of the genetic defect contained factor VIII exons 16-24 coding sequences, a spacer sequence, a branch point, a polypyrimidine tract, and a 3' acceptor splice (Figure 44A). The successful correction of the mutated DNA encoding factor VIII utilizing a trans-splicing reaction further supports the general application of PTMs for correction of genetic defects.

The methods and compositions of the invention may also be used to regulate gene expression in plants. For example, trans-splicing may be used to place COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:49 FROM- T-966 P038/099 F-541 31 00 0 0 the expression of any engineered gene under the natural regulation of a chosen target plant gene, thereby regulating the expression of the engineered gene. Tans-splicing may also be used to prevent the expression of engineered genes in non-host plants or to convert an endogenous gene product into a more desirable product In a specific embodiment of the invention tran-splicing may be used to regulate the expression of the insecticidal gene that produces Bt toxin (Bacillus Sthuringiensis). For example, the PTM maybe designed to trans-splice into an injury response gene (pre-mRNA) that is expressed only after an insect bites the plant.

O Thus, all cells of the plant would carry the gene for Bt in the PTM, but the cells would CN 10 only produce Bt when and where an insect injures the plant. The rest of the plant will make little or no Bt. A PTM could tras-splice the Bt gene into any chosen gene with a desired pattern of expression. Further, it should be possible to target a PTM so that no Bt is produced in the edible portion of the plant.

One advantage associated with the use of PTMs is that the PTM acquires the native gene control elements of the target gene, thus, reducing the time and effort that might otherwise be spent attempting to identify and reconstitute appropriate regulatory sequences upstream of an engineered gene. Thus, expression of the PTM regulated gene should occur only in those plant cells containing the target pre-mRNA. By targeting a gene not expressed in the edible portion of the plant or in the pollen, trans-splicing can alleviate opposition to genetically modified plants, as consumers would not be eating the proteins made from modified genes. The edible portion of such crops should test negative for genetically modified proteins.

In addition, PTM can be targeted to a unique sequence of the host gene that is not present in other plants. Therefore, even if the gene (DNA) which encodes the PTM jumps to another species of plant, the PTM gene will not have an appropriate target for trans-splicing. Thus, trats-splicing offers a "fail-safe" mode for prevention of gene "jumping" to other plant species: the PTM gene will be expressed only in the engineered host plant, which contains the appropriate target pre-mRNA. Expression in non-engineered plants would not be possible.

Trans-splicing also provides a more efficient way to convert one gene product into another. For example, trans-splicing ribozymes and chimeric oligos can COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:49 FROM- T-966 P039/099 F-541 32 00 0 0 be incorporated into corn genomes to modify the ratio of saturated to unsaturated oils.

Trans-splicing can also be used to convert one gene product into another.

Various delivery systems are known and can be used to transfer the compositions of the invention into cells, e.g. encapsulation in liposomes, micropartioles, microcapsules, recombinant cells capable of expressing the composition, receptor-mediated endocytosis (see, Wu and Wu, 1987, J. Biol.

4Chem. 262:4429-4432), construction of a nucleic acid as part of a retroviral or other c vector, injection ofDNA, electroporation, calcium phosphate mediated transfection, o etc.

C' 10 The compositions and methods can be used to treat cancer and other serious viral infections, autoimmune disorders, and other pathological conditions in which the alteration or elimination of a specific cell type would be beneficial.

Additionally, the compositions and methods may also be used to provide a gene encoding a functional biologically active molecule to cells of an individual with an inherited genetic disorder where expression of the missing or mutant gene product produces a normal phenotype.

In a preferred embodiment, nucleic acids comprising a sequence encoding a PTM are administered to promote PTM function, by way of gene delivery and expression into a host cell. In this embodiment of the invention, the nucleic acid mediates an effect by promoting PTM production. Any of the methods for gene delivery into a host cell available in the art can be used according to the present invention. For general reviews of the methods of gene delivery see Strauss, M. and Barranger, 1997, Concepts in Gene Therapy, by Walter do Gruyter Co., Berlin; Goldspiel et 1993, Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 33:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev.

Biochem. 62:191-217; 1993, TIBTECH 11(5):155-215. Exemplary methods are described below.

Delivery of the nucleic acid into a host cell may be either direct, in which case the host is directly exposed to the nucleic acid or nucleic acid-carrying vector, or indirect, in which case, host cells are first transformed with the nucleic acid COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:49 FROM- T-966 P040/099 F-541 33 00 0 0 C) in vitro, then transplanted into the host. These two approaches are known, respectively, as in vivo or ex vivo gene delivery.

00 In a specific embodiment, the nucleic acid is directly administered in vivo, where it is expressed to produce the PTM. This can be accomplished by any of numerous methods known in the art, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, eg. by infection using a defective or attenuated retroviral or other viral vector (see U.S. Patent No. 4,980,286), or by direct injection of naked DNA, or by use of O microparticle bombardment a gene gun; Biolistic, Dupont), or coating with CN 10 lipids or cell-surface receptors or transfecting agents, encapsulation in liposomes, microparticles, or microcapsules, or by administering it in linkage to a peptide which is known to enter the nucleus, by administering it in linkage to a ligand subject to receptor-mediated endocytosis (see Wu and Wu, 1987, J. Biol. Chem. 262:4429- 4432).

In a specific embodiment, a viral vector that contains the PTM can be used. For example, a retroviral vector can be utilized that has been modified to delete retroviral sequences that are not necessary for packaging of the viral genome and integration into host cell DNA (see Miller et al., 1993, Moth. Enzymol. 217:581-599).

Alternatively, adenoviral or adeno-associated viral vectors can be used for gene delivery to cells or tissues. (See, Kozarsky and Wilson, 1993, Current Opinion in Genetics and Development 3:499-503 for a review of adenovirus-based gene delivery).

Another approach to gene delivery into a cell involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection. Usually, the method of transfer includes the transfer of a selectable marker to the cells. The cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene. The resulting recombinant cells can be delivered to a host by various methods known in the art. In a preferred embodiment, the cell used for gene delivery is autologous to the host cell.

COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:50 FROM- T-966 P041/99 F-5,41 34 00 0 r The present invention also provides for pharmaceutical compositions 00 comprising an effective amount of a PTM or a nucleic acid encoding a PTM, and a pharmaceutically acceptable carrier. In a specific embodiment, the term "phannaceutically acceptable" means approved by a regulatory agency of the Federal I 5 or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is C- administered. Examples of suitable pharmaceutical carriers are described in 0 "Remington's Pharmaceutical sciences" by E.W. Martin.

C 10 In specific embodiments, pharmaceutical compositions are administered: in diseases or disorders involving an absence or decreased (relative to normal or desired) level of an endogenous protein or function, for example, in hosts where the protein is lacking, genetically defective, biologically inactive or underactive, or under expressed; or in diseases or disorders wherein, in vitro or in vivo, assays indicate the utility of PTMs that inhibit the function of a particular protein. The activity of the protein encoded for by the chimeric mRNA resulting from the PTM mediated trans-splicing reaciion can be readily detected, by obtaining a host tissue sample from biopsy tissue) and assaying it in vitro for mRNA or protein levels, structure and/or activity of the expressed chimeric mRNA.

Many methods standard in the art can be thus employed, including but not limited to immunoassays to detect and/or visualize the protein encoded for by the chimeric mRNA Western blot, immunoprecipitation followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunocytochemistry, etc.) and/or hybridization assays to detect formation of chimeric mRNA expression by detecting and/or visualizing the presence of chimeric mRNA Northern assays, dot blots, in situ hybridization, and Roverse-Transcription PCR, etc.), etc.

The present invention also provides for pharmaceutical compositions comprising an effective amount of a PTM or a nucleic acid encoding a PTM, and a pharmaceutically acceptable carrier. In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:50 FROM- T-966 P042/099 F-541 00 0 0 pharmacopeia for use in animals, and more particularly in humans, The term "carrier" refers to a diluent, adjuvant, exipient, or vehicle with which the therapeutic is 00 administered. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical sciences" by E.W. Martin. In a specific embodiment, it may be desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment. This maybe achieved by, for example, and Snot by way of limitation, local infusion during surgery, topical application, in conjunction with a wound dressing after surgery, by injection, by means of a 0 catheter, by means of a suppository, or by means of an implant, said implant being of C- 10 a porous, non-porous, or gelatinous material, including membranes, such as sialastio membranes, or fibers. Other control release drug delivery systems, such as nanoparticles, matrices such as controlled-release polymers, hydrogels.

The PTM will be administered in amounts which are effective to produce the desired effect in the targeted cell. Effective dosages of the PTMs can be determined through procedures well known to those in the art which address such parameters as biological half-life, bioavailability and toxicity. The amount of the composition of the invention which will be effective will depend on the nature of the disease or disorder being treated, and can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges.

The present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

5.3.2. USE OF PTM MOLECULES FOR EXON TAGGING In view of current efforts to sequence and characterize the genomes of humans and other organisms, there is a need for methods that facilitate such COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:51 FROM- T-966 P043/099 F-541 36 00 0 0 characterization. A majority of the informationi currntly obtained by genomic 00 mapping and sequencing is derived from complementary DNA (cDNA) libraries, 00 which are made by reverse transcription of mRNA into cDNA. Unfortunately, this process causes the loss of information concering intron sequences and the location of exon/intron boundaries.

The present invention encompasses a method for mapping exon-intron boundaries in pre-mRNA molecules comprising contacting a pre-trans-splicing molecule with a pre-mRNA molecule under conditions in which a portion of the pre- 0 trans-splicing molecule is trans-spliced to a portion of the target pre-mRNA to form a c-i 10 chimeric mRNA; (ii) amplifying the chimeric mRNA molecule; (iii) selectively purifying the amplified molecule; and (iv) determining the nucleotide sequence of the amplified molecule thereby identifying the intron-exon boundaries.

In an embodiment of the present invention, PTMs can be used in transsplicing reactions to locate exon-intron boundaries in pre-mRNAs molecules. PTMs for use in mapping ofintron-exon boundaries have structures similar to those i described above in Section 5.1. Specifically, the PTMs contain a target binding domain that is designed to bind to many pre-mRNAs: (ii) a 3' splice region that includes a branch point, pyrimidine tract and a 3' splice acceptor site, or a 5' splice donor site; (iii) a spacer region that separates the mRNA splice site from the target binding domain; and (iv) a tag region that will be trans-spliced onto a pre-mRNA.

Alternatively, the PTMs to be used to locate exon-intron boundaries may be engineered to contain no target binding domain.

For purposes of intron-exon mapping, the PTMs are genetically engineered to contain target binding domains comprising random nucleotide sequences. The random nucleotide sequences contain at least 15-30 and up to several hundred nucleotide sequences capable of binding and anchoring apre-mRNA so that the spliceosome processing machinery of the nucleus can trans-splice a portion (tag or marker region) of the PTM to a portion of the pre-mRNA. PTMs containing short target binding domains, or containing inosines bind under less stringent conditions to the pre-mRNA molecules. In addition, strong branch point sequences and pyrimidine tracts serve to increase the non-specificity of PTM trans-splicing.

COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:51 FROM- T-966 P044/099 F-541 37 00 0 0 The random nucleotide sequences used as target binding domains in the PTM molecules can be generated using a variety of different methods, including, 00 but not limited to, partial digestion of DNA with restriction endonucleases or mechanical shearing of the DNA. The use of such random nuclootide sequences is O' 5 designed to generate a vast array of PTM molecules with different binding activities for each target pre-mRNA expressed in a cell. Randomized libraries of oligonucleotides can be synthesized with appropriate restriction endonucleases crecognition sites on each end for cloning into PTM molecules genetically engineered O into plasmid vectors. When the randomized oligonucleotides are litigated and 10 expressed, a randomized binding library of PTMs is generated.

In a specific embodiment of the invention, an expression library encoding PTM molecules containing target binding domains comprising random nucleotide sequences can be generated using a variety of methods which are well known to those of skill in the art. Ideally, the library is complex enough to contain PTM molecules capable of interacting with each target pre-mRNA expressed in a cell.

By way of example, Figure 9 is a schematic representation of two forms of PTMs which can be utilized to map intron-exon boundaries. The PTM on the left is capable of non-specifically trans-splicing into a pre-mRNA 3' splice site, while the PTM on the right is capable of trans-splicing into a pre-mRNA 5' splice site.

Trans-splicing between the PTM and the target pre-mRNA results in the production of a chimeric mRNA molecule having a specific nucleotide sequence "tag" on either the 3' or 5' end of an authentic exon.

Following selective purification, a DNA sequencing reaction is then performed using a primer which begins in the tag nucleotide sequence of the PTM and proceeds into the sequence of the tagged exon. The sequence immediately following the last nucleotide of the tag nucleotide sequence represents an exon boundary. For identification of intron-exon tags, the trans-splicing reactions of the invention can be performed either in vitro or in vivo using methods well known to those of skill in the art.

COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:51 FROM- T-966 P045/099 F-541 38 00 0O 0 ci L) 5.3.3. USE OF PTM MOLECULES FOR IDENTIFICATION C OF PROTEINS EPRSSED IN A CELL 00 In yet another embodiment of the invention, PTM mediated transsplicing reactions can be used to identify previously undetected and unknown proteins 5 expressed in a cell. This method is especially useful for identification of proteins that O cannot be detected by a two-dimensional electrophoresis, or by other methods, due to inter alia the small size of the protein, low concentration of the protein, or failure to c detect the protein due to similar migration patterns with other proteins in twoo dimensional clectrophoresis.

The present invention relates to a method for identifying proteins expressed in a cell comprising contacting a pre-trans-splicing molecule containing a random target binding domain and a nuoleotide sequence encoding apeptide tag with a pre-mRNA molecule under conditions in which a portion of the pre-transsplicing molecule is trans-spliced to a portion of the target pre-mRNA to form a chimeric mRNA encoding a fusion polypeptide or separating it by gel electrophoresis (ii) affinity purifying the fusion polypeptide; and (iii) determining the amino acid sequence of the fusion protein.

To identify proteins expressed in a cell, the PTMs of the invention are genetically engineered to contain: a target binding domain comprising randomized nucleotide sequences; (ii) a 3' splice region that includes a branch point, pyrimidine tract and a 3' splice acceptor site and/or a 5' splice donor site; (iii) a spacer region that separates the PTM splice site from the target binding domain; and (iv) nucleotide sequences encoding a marker or peptide affinity purification tag. Such peptide tags include, but are not limited to, HIS tags (6 histidine consecutive residues) (Janknecht, et al., 1991 Proc. Natl. Acad. ScL USA 88:8972-8976), glutathione-S-transferase (GST) (Smith, D.B. and Johnson 1988, Gene 67:31) (Pharmacia) or FLAG (Kodak/IBI) tags (Nisson, J. et aL J. Mol. Recognit., 1996, 5:585-594).

Trans-splicing reactions using such PTMs results in the generation of chimeric mRNA molecules encoding fusion proteins comprising protein sequences normally expressed in a cell linked to a marker or peptide affinity purification tag.

The desired goal of such a method is that every protein synthesized in a cell receives a COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:51 FROM- T-966 P046/099 F-541 39 00 0 0 ci C) marker or peptide affinity tag thereby providing a method for identifying each protein Sexpressed in a cell.

00 In a specific embodiment of the invention, PTM expression libraries encoding PTMs having different target binding domains comprising random nucleotide sequences are generated. The desired goal is to create a PTM expression library that is complex enough to produce a PTM capable of binding to each pre- SmRNA expressed in a cell. In a preferred embodiment, the library is cloned into a C imammalian expression vector that results in one, or at most, a few vectors being O present in any one cell.

Ci 10 To identify the expression of chimeric proteins, host cells are transformed with the PTM library and plated so that individual colonies containing one PTM vector can be grown and purified. Single colonies are selected, isolated, and propagated in the appropriate media and the labeled chimeric protein exon(s) fragments are separated away from other cellular proteins using, for example, an affinity purification tag. For example, affinity chromatography can involve the use of antibodies that specifically bind to a peptide tag such as the FLAG tag. Alternatively, when utilizing HIS tags, the fusion proteins are purified using a Ni 2 nitriloacetic acid agarose columns, which allows selective elution of bound peptide eluted with imidazole containing buffers. When using GST tags, the fusion proteins are purified using glutathione-S-transferase agarose beads. The fusion proteins can then be eluted in the presence of free glutathione.

Following purification of the chimeric protein, an analysis is carried out to determine the amino acid sequence of the fusion protein. The amino acid sequence of the fusion protein is determined using techniques well known to those of skill in the art, such as Edman Degradation followed by amino acid analysis using HPLC, mass spectrometry or an amino acid analyzation. Once identified, the peptide sequence is compared to those sequences available in protein databases, such as GenBank. If the partial peptide sequence is already known, no further analysis is done. If the partial protein sequence is unknown, then a more complete sequence of that protein can be carried out to determine the full protein sequence. Since the fusion protein will contain only a portion of the full length protein, a nucleic acid COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-' 08 14:51 FROM- T-966 P047/099 F-541 00 encoding the full length protein can be isolated using conventional methods. For 00 example, based on the partial protein sequence oligonucleotide primers can be generated for use as probes or PCR primers to screen a oDNAlibrary.

6. EXAMPLE: PRODUCTION OF RANS-SPLICING

MOLECULES

The following section describes the production of PTMs and the demonstration that such molecules are capable of mediating trais-splicing reactions resulting in the production of chimeric mRNA molecules.

Ci 6.1. MATERALS AND METHODS 6.1.1. CONSTRUCTION OF PRE-mnRNA MOLECULES Plasmids containing the wild type diphtheria toxin subunit A (DT-A, wild-type accession #K01722) and a DT-A mutant (CRM 197, no enzymatic activity) were obtained from Dr. Virginia Johnson, Food and Drug Administration, Bethesda, Maryland (Uchida et al., 1973 1. Biol. Chem 248:3838). For in vitro experiments, DT-A was amplified using primers: DT-1F GGCGCTGCAGGGCGCTGATGATGTTGTTG); and DT-2R (S'-GGCGAAG CTTGGATCCGACACGATTTCCTGCACAG out with PstI and Hind, and cloned into PatI and Hindmli digested pBS(-) vector (Stratagene, La Jolla, CA). The resulting clone, pDTA was used to construct the individual PTMs. pPTM+: Targeted construct. Created by inserting IN3-1

CACCCGGGCCTGACTCGAGTACTAACTGGTACCCTTCTTCCTOCA

and IN2-4 CCCGGGTGAAGCATCTAGAG) primers into EcoRI and Pstl digested pDTA.

pPTM+Sp: As pPTM+ but with a 30 bp spacer sequence between the BD and BP.

Created by digesting pPTM+ with Xhol and ligating in the oligonucleotides, spacer S (5-TCGAGCAACGTTATAATAATGTTC) and spacer AS TCGAGAACATTATT ATAACGTTGC). For in vivo studies, an EcoRI and HindII fragment of pcPTM+Sp was cloned into mammalian expression vector pcDNA3.1 (Invitrogen), under the control of a CMV promoter. Also, the methioine at codon 14 was changed into isoleucine to prevent initiation of translation. The resulting COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:52 FROM- T-966 P048/099 F-541 1 41 plasmid was designated as pcPTMSp. pPTM+CRM! As pPTM-Sp but the wild type DT-A was substituted with CRM mutant DT-A Uchida, et at., 1973, J. Biol.

Che=. 248:3838). This was created by PCR amplification of a DT-A mutant (mutation at 052E) using primers DT-IF and DT-2R. For in va studies, an ECoR! S Hind! fragment ofPTM+CRM was cloned into pc3. IDNA that resulted in pcPTM+ARM. PTM-: Non-targeted construct. Created by digestion of PTh(+ with EcoRi and Pat L gel purified to remove the binding domain followed by ligation of the oligonucleotides, IN-S CGAO) and IN-6 (5'-TGCTTCACCC GIGCCTGATCTAGAG). PTM-Sp, is an identical version of the PTM-, except it has a 30 bp spacer sequence at the Pstil site.

Similarly, the splice mutants and and safety variants [PTM4-SF-Pyl, PTM+SF-Py2. PTM+SFBP3 and PTM4SFBP3-Pyl] were constructed either by insertion or deletion of specific sequences (see 1).

Table 1. Binding/non-binding domain, 3P, PPT and 3' as sequences of different PTMs.

PTM canshuct BDNBD J PPT 23's PTM+S (targeted) :TGCTCACCCirCCrOA TACTAC CTCMLI MT1T1 f CAG PTM-Sp (non-twaeted) ±CAACGrATAATAATGTr TACTAAC C CTLI mTILL CAG 1Th4+Py ;TGC 7CACCGOCUJ=A GCTOAT aTGATrAATAGCOO ACO PTM4y-)AG(-) :TGCTTCACCCGGGCrGA TACTAAC 0CrGGACGCOAAOr ACO f+SF :CTGGGACAAGOACACTGCTT CACCC3GTAGTAGACCACA GXCCCTAAGCC TACTAAC CTTCTGITTTrCTC CAG PF+SF-PY :As i Ph+SF TACTAAC CrTfrGT1A 1TOTC CAO PfA+SF-Py :As in PTM+SF TACTAAC OTTCTOTCCnOeTCTC CAG PTM+SF-BP3 :As in PTM+SF TOCTOAC C1WTGflTlTFTC CAD PTM+sPB13-Py As in PThi+SF OTGOAG CTTCTGTArrATCTC CAw0 Nucleotides in bold indicate the mutations compared to normal BP, PPT and 3' splice site.

Branch site A is underlined. The nucleotides in italics indicates the mismatch introduced into safety BD to mask the BP sequence in the rIM.

COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:52 FROM- T-966 P49/099 F-541 42 00 0 0 9 A double-trans-splicing PTM construct (DS-PTM) was also made 00 adding a 5' splice site and a second target binding domain complementary to the second intron of pHCG pro-mRNA to the 3' end of the toxin coding sequence of PTM+SF (Figure A).

6.1.2. 6HCG6 TARGET PRE-mRNA To produce the in vitro target pre-mRNA, a SacI fragment of HCG c *gene 6 (accession #X00266) was cloned into This produced an 805 bp insert o from nucleotide 460 to 1265, which includes the 5' untranslated region, initiation codon, exon 1, intron 1, exon 2, and most ofintron 2. For in vive studies, an EcoRI and BamHI fragment was cloned into mammalian expression vector (pc3.1DNA), producing pHCG6.

6.1.3. mRNA PREPARATION For in vitro splicing experiments, pHCG6, P-globin pre-mRNA and different PTM mRNAs were synthesized by in vitro transcription of BamHI and HindHI digested plasmid DNAs respectively, using T7 mRNA polymerase (Pasman Garcia-Blanco, 1996, Nucleic Acids Res. 24:1638). Synthesized mRNAs were purified by electrophoresis on a denaturing polyacrylamide gel, and the products were excised and eluted.

6.1.4 IN VITRO SPLICING PTMs and target pre-mRNA were annealed by heating at 98°C followed by slow cooling to 30-34'C. Each reaction contained 4 pt of annealed mRNA complex (100 ug of target and 200 ng of PTM), 1X splice buffer (2 mM MgC12, 1 mM ATP, 5 mM creatinine phosphate, and 40 mM KCI) and 4 Pl of HeLa splice nuclear extract (Promega) in a 12.5 pl final volumae. Reactions were incubated at 30"C for the indicated times and stopped by the addition of an equal volume of high salt buffer (7 M urea, 5% SDS, 100 mM LiC, 10 mM EDTA and 10 mM TrisHCI, pH Nucleic acids were purified by extraction with phenol:chloroform:isoamyl alcohol (50:49:1) followed by ethanol precipitation.

COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:52 FROM- T-966 P050/099 F-541 43 00 0 S6.1.5. REVEtRSE TRANSCRIPTION-PCR REACTIONS 00 RT-PCR analysis was performed using EZ-RT PCR kit (Perkin-Elhner, Foster City, CA). Each reaction contained 10 ng of cis- or trans-spliced mRNA, or 1-2 pg of total mRNA, 0.1 pl of each 3' and 5' specific primer, 0.3 mM of each dNTP, iX EZ buffer (50 mM bicine, 115 mM potassium acetate, 4% glycerol, pa mM magnesium acetate and 5 U of rflh DNA polymerase in a 50 I reaction volbnme. Reverse transcription was performed at 60C for 45 min followed by PCR N amplification of the resulting cDNA as follows: one cycle of initial denaturation at o 94C for 30 see, and 25 cycles of denaturation at 94'C for 18 sec and annealing and extension at 600C for 40 see, followed by a 7 min final extension at 70*C. Reaction products were separated by electrophoresis in agarose gels.

Primers used in the study were as follows: DT-1F: GGCGCTGCAGGGCGCTGATGATGTTGTTG DT-2R: GGCGAAGCTGGATCCGACACGATTTCCTGCACAGG DT-3R: CATCGTCATAATTTCCTTGTG DT-4R: ATGGAATCTACATAACCAGG DT-SR: GAAGGCTGAGCACTACACGC HCG-R2: CGGCACCGTGGCCGAAGTGG, Bio-HCG-F: ACCGGAATTCATGAAGCCAGGTACACCAGG n-globulin-F: (3GGCAAGGTGAACGTGGATG ft-globulin-R ATCAGGAGTGGACAGATCC 6.1.6. CELL GROQWTHL TRANSFECTION AND mRNA

ISOLATION

Human lung cancer cell line 11299 (ATCC accession CRL-5803) was grown in RPMI medium supplemented with 10% fetal bovine serum at 371C in a C02 environment. Cells were transfected with pcSp+CRM (CRM is a nonfunctional toxin), a vector expressing a PTM, or vector alone (pcDNA3.1) using lipofectamine reagent (Life Tcbnologies, Gaithersburg, MD). The assay was scored for neomycin resistance (neo) colony formation two weeks after transfection. Four neo colonies were selected and expanded under continued neo selection. Total COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:52 FROM- T-96;6 P051/099 F-541 44 00 0 0 cellular mRNA was isolated using RNA exol (BioChain Institute, Inc., San Leandro, 00 CA) and used for RT-PCR.

6.1.7. TRAS-SPLICING IN TUMORS IN NUDE MICE Eleven nude mice were bilaterally injected (except B10, B11 and B12 had 1 tumor) into the dorsal flank subcutaneous space with 1 x 10 1 11299 human lung tumor cells (day On day 14, the mice were given an appropriate dose of anesthesia and injected with, or without electroporation (T820, BTX Inc., San Diego, O CA) in several orientations with a total volume of 100 1A of saline containing 100 jig C' pcSp+CRM with or without pcpHCG6 or pcPTM+Sp. Solutiohs injected into the right side tumors also contained India ink to mark needle tracks. The animals were sacrificed 48 hours later and the tumor excised and immediately frozen at -80 For analysis, 10 mg of each tumor was homogenized and mRNA was isolated using a Dynabeads mRNA direct kit (Dynal) following the manufacturers directions. Purified mRNA (2 p1 of 10 lA total volume) was subjected to RT-PCR using PHCG-F and DT- 5R primers as described earlier. All samples were re-amplified using DT-3R, a nested DT-A primer and biotinylated DHCG-F and the products were analyzed by electrophoresis on a 2% agarose gel. Samples that produced a band were processed into single stranded DNA using M280 Streptavidin Dynabeads and sequenced using a toxin specific primer (DT-3R).

6.2. RESULTS 6.2.1. SYNTHESIS OF PTM A prototypical trans-splicing mRNA molecule, pcPTM+Sp (Figure IA) was constructed that included: an 18 nt target binding domain (complementary to PHCG6 intron a 30 nucleotide spacer region, branch point (BP) sequence, a polypyrimidine tract (PPT) and an AG dinucleotide at the 3' splice site immediately upstream of an exon encoding diphtheria toxin subunit A (DT-A) (Uchida et al., 1973, J. Biol. Chem. 248:3838). Later DT-A exons were modified to eliminate translation initiation sites at codon 14. The PTM constructs were designed for maximal activity COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:52 FROM- T-966 P052/099 F-541 00 0 0 r1 in order to demonstrate trans-splicing; therefore, they included potent 3' splice 00 elements (yeast BP and a mamIalian PPT) (Moore et at, 1993, In The mRNA World, RF. Gesteland and L.F. Atkins, eds. (Cold Spring Harbor, Now York: Cold Spring Harbor Laboratory Press). PHCG6 pre-mRNA (Talmadge et al, 1984, I 5 Nucleic Acids Res. 12:8415) was chosen as a model target as this gene is expressed in most tumor cells. It is not expressed in normal adult cells, with the exception of some "1 in the pituitary gland and gonads. (Acevedo et al., 1992, Cancer 76:1467; Hoon et al., 1996, Int J. Cancer 69:369; Bellet at al, 1997, Cancer Res. 57:516). As shown in o Figure 1C, pcPTM+Sp forms conventional Watson-Crick base pairs by its binding C' 10 domain with the3' end of pHCG6 intrn 1, masking the intonic 3' splice signals of the target. This feature is designed to facilitate trans-splicing between the target and the PTM HeLa nuclear extracts were used in conjunction with established splicing procedures (Pasman Garoia-Blanco, 1996, Nucleic Acids Res. 24:1638) to test if a PTM construct could invade the PHCG6 pre-mRNA target. The products of in itro trans-splicing were detected by RT-PCR, sing primers specific for chimeric mRNA molecules. The predicted product of a successful trans-splicing reaction is a chimeric mRNA comprising the first exon of pHCG6, followed immediately by the exon contributed from pcPTM+Sp encoding DT-A (Figure 1C). Such chimeric mRNAs were readily detected by RT-PCR using primers pHCG-F (specific to PHCG6 exon 1) and DT-3R (specific to DT-A, Figure 2A, lanes At time zero or in the absence of ATP, no 466 bp product was observed, indicating that this reaction was both ATP and time dependent.

The target binding domain ofpcPTM+Sp contained 18 nucleotides complementary to pHCG6 intron 1 pre-mRNA and demonstrated efficient transsplicing (Figure 2A, lanes Tans-splicing efficiency decreased at least 8 fold (Figure 2, lanes 3-4) using non-targeted PTM-Sp, which contains a noncompleimetary 18 nucleotide "non-binding domain". prans-splicing efficiencies of PTM mRNAs with or without a spacer between the binding domain and BP were also compared. This experiment demonstrated a significant increase in the efficiency of trans-splicing by the addition of a spacer (Figure 2B, lanes 2 To facilitate the COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:53 FROM- T-966 P053/09 F-541 46 00 0 0 recruitment of splicing factors required for efficient trans-splicing, some space may be needed between the 3' splice site and the double-stranded secondary structure 00 produced by the binding domain/target interaction.

To investigate the effect of PTM length on trans-splicing specificity, shorter PTMs were synthesized from AccI cut PTM plasmid (see Figure This N\ eliminated 479 nt from the 3' end of the DT-A coding sequence. Figure 2B shows the trans-splicng ability of a targeted short PTM(+) (lanes 10-12), compared to a nontargeted short PTM(-) (lanes 14-17). Short PTM+ produced substantially more trans- O spliced product (Figure 2B, lane 12) than its counterpart, non-targeted short PTM C, 10 (Figure 2B, lane 17). These experiments indicate that longer PTMs may have increased potential to mediate trwns-splicing non-specifically.

6.2.2. ACCURACY OF PTM SPLICEOSOME MEDIATED TRANS-SPLICING To confirm that trins-splicing between the pcPTM+Sp and pHCG6 target is precise, RT-PCR amplified product was produced using 5' biotinylated pHCG-F and nonbiotinylated DT-3R primers. This product was converted into single stranded DNA and sequenced directly with primer DT-3R (DT-A specific reverse primer) using the method of Mitchell and Merril (1989, Anal. Biochem. 178:239).

Trans-splicing occurred exactly between the predicted splice sites (Figure 3), confirming that a conventional pre-riRNA can be invaded by an engineered PTM construct during splicing; moreover, this reaction is precise.

In addition selective trans-splicing of a double splicing PTM (DS- PTM) was observed (Figure SB). The DS-PTM can produce trans-splicing by contributing either a 3' or 5' splice site. Further, DS-PTMs canbe constructed which will be capable of simultaneously double-trans-splicing, at both a 3' and 5' site, thereby permitting exon replacement. Figure SB demonstrates that in this construct the 5' splice site is most active at a 1:1 concentration of target PHCG pre-mRNA:DS- PTM. At a 1:6 ratio the 3' splice site is more active.

COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:53 FROM- T-966 P054/099 F-541 47 00 0 0 6.2.3. 3' SPLICE SITES ARE ESSENTIAL FOR PTM TRANS-SPLICING 00 In general, the 3' splice site contains three elements: 1) a BP sequence located 5' of the acceptor site, 2) a PPT consisting of a short run of pyrimidine residues, and 3) a YAG trinucleotide splice site acceptor at the intron-exon border (Senapathy et al., 1990, Cell 91:875; Moore at al, 1993). Deletion or alteration of O one of these sequence elements are known to either decrease or abolish splicing (Aebi et al., 1986; Reed Maniatis 1988, Genes Dev. 2:1268; Reed, 1989, Genes Dev.

(C 3:2113; Roscigno et al., 1993, J. Biol. Chem. 268:11222; Coolidge et al., 1997, o Nucleic Acids Res. 25:888). The role of these conserved elements in targeted transsplicing was addressed experimentally. In one case all three cis elements (BP, PPT and AG dinucleotide) were replaced by random sequences. A second splicing mutant was constructed in which the PPT and the 3' splice site acceptor were mutated and substituted by random sequences. Neither construct was able to support trans-splicing in vitro (Figure 2A, lanes suggesting that, as in the case of conventional cit-splicing, the PTM trans-splicing process also requires a functional BP, PPT and AG acceptor at the 3' splice site.

6.2.4. DEVELOPMENT OF A "SAFETY" SPLICE SITE TO INCREASE SPECIFICITY To improve the levels of target specificity achieved by the inclusion of a binding domain or by shortening the PTM, the target-binding domain of several PTM constructs was modified to create an intra-molecular stem to mask the 3' splice site (termed a "safety PTM"). The safety stem is formed by portions of the binding domain thatpartially base pair with regions of the PTM 3' splice site or sequences adjacent to them, thereby blocking the access of spliceosomal components to the PTM 3' splice site prior to target acquisition (Figure 4A, PTM+SF). Base pairing between free portions of the PTM binding domain and pHCG6 target region unwinds the safety stem, allowing splicing factors such as U2AF to bind to the PTM 3' splice site and initiate trans-splicing (Figure 4B).

This concept was tested in splicing reactions containing either PTM+SF (safety) or pcPTM+Sp (linear), and both target (pHCG6) and non-target (P- COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:53 FROM- T-966 P055/099 F-541 48 00

O

O

ci globin) pre-mRNA. The spliced products were subsequently analyzed by RT-PCR 00 and gel electrophoresis. Using PHCG-F and DT-3R primers, the specific 196 bp trans-spliced band was demonstrated in reactions containing pHCG target and either linear PTM (pcPTM+Sp, Figure 5, lane 2) or safety PTM (PTM+SF, Figure 5, lane 8).

Comparison of the targeted trans-splicing between linear PTM (Figure 5, lane 2) and \O safety PTM (Figure 5, lane 8) demonstrated that the safety PTM trans-spliced less.

efficiently than the linear PTM.

Ci Non-targeted reactions were amplified using 0-globin-F (specific to

O

0 exon 1 of p.globin) and DT-3R primers. The predicted product generated by nonspecific PTM tran-splicing with p-globin pre-mRNA is 189 bp. Non-specific transsplicing was evident between linear PTM and P-globin pre-mRNA (Figure 5, lane In contrast, non-specific trans-splicing was virtually eliminated by the use of safety PTM (Figure 5, lane 11). This was not unexpected, since the linear PTM was designed for maximal activity to prove the concept of spliceosome-mediated transsplicing. The open structure of the linear PTM combined with its potent 3' splice sites strongly promotes the binding of splicing factors. Once bound, these splicing factors can potentially initiate trans-splicing with any 5' splice site, in a process similar to trans-splicing in trypanosomes. The safety stem was designed to prevent splicing factors, such as U2AF from binding to the PTM prior to target acquisition. This result is consistent with a model that base-pairing between the free portion of the binding domain and the pHCG6 target unwinds the safety stem (by mRNA-mRNA interaction), uncoveing the 3' splice site, permitting the recruitment of splicing factors and initiation of trns-splicing. No trans-splicing was detected between Pglobin and pHCG6 pre-mRNAs (Figure 5, lanes 3, 6, 9 and 12).

6.2.5. IN VTRO TRANS-SPLICING OF SAFETY PTM AND VARIANTS To better understand the role of ds-elements at the 3' splice site in trans-splicing a series of safety PTM variants were constructed in which either the PPT was weakened by substitution with purines and/or the BP was modified by base substitution (see Table In vitro trans-splicing efficiency of the safety (PTM+SF) was compared to three safety variants, which demonstrated a decreased ability to COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:53 FROM- T-966 P056/099 F-541 49 00 0 0 Dtrans-splice. The greatest effect was observed with variant 2 (PTM+SFPy2), which 00 was trans-splicing incompetent (Figure 40, lanes This inhibition of transsplicing may be attributed to a weakened PPT and/or the higher Tm of the safety stem.

In contrast, variations in the BP sequence (PTM+SFBP3) did not markedly effect trans-splicing (Figure 4C, lanes This was not surprising since the modifications 0 introduced were Within the mammalian branch point consensus range YNYURAC (where Y pyrimidine, R purine and N= any nuclcotide) (Moore e al., 1993).

This finding indicates that the branch point sequence can be removed without O affecting splicing efficiency. Alterations in the PPT (PTM+SF-Pyl) decreased the Ci 10 level of trans-splicing (lanes Similarly, when both BP and PPT were altered PTM+SFBP3-Pyl, they caused a further reduction in trans-splicing (Figure 4C, lanes 9-10). The order of rans-splicing efficiency ofthese-safety variants is PTM+SF>PTMtSFBP3> PTM+SFPyl>PTM:SFBP3-Pyl>PTM+SFPy2. These results confirm that both the PPT and BP are important for efficient in vitro transsplicing (Roscigno et al., 1993, J. Biol. Chem. 268:11222).

6.2.6. COMPETITION BETWEEN CIS- AND TRANS- SPLICING To determine if it was possible to block pre-mRNA cis-splicing by increasing concentrations of PTM, experiments were performed to drive the reaction towards tran-splicing. Splicing reactions were conducted with a constant amount of PHCG6 pre-mRNA target and various concentrations of trans-splicing PTM. Cissplicing was monitored by RT-PCR using primers to pHCG-F (exon 1) and PHCG-R2 (exon This amplified the expected 125 bp cis-spliced and 478 bp unspliced products (Figure 6A). The primers pHCG-F and DT-3R were used to dtect transspliced products (Figure 6B). At lower concentrations of PTM, cis-splicing (Fig. 6A, lanes 1-4) predominated over trans-splicing (Figure 6B, lanes Cis-splicing was reduced approximately by 50% at a PTM concentration 1.5 fold greater than target.

Increasing the PTM mRNA concentration to 3 fold that of target inhibited cis-splicing by more than 90% (Figure 6A, lanes with a concomitant increase in the transspliced product (Figure 63, lanes 6-10). A competitive RT-PCR was performed to simultaneously amplify both cis and trans-spliced products by including all three COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:54 FROM- T-966 P057/099 F-541 00 0 0 primers (pHCG-F, HCG-R2 and DT-3R) in a single reaction. This experiment had 00 similar results to those seen in Figure 6, demonstrating that under in vitro conditions, a PTM can effectively block target pre-mRNA cis-splicing and replace it with the production of an engineered trans-spliced chimeric mRNA.

6.2.7. TRANS-SPLICING IN TISSUE CULTURB To demonstrate the mechanism of trans-splicing in a cell culture model, the human lung cancer line H1299 (pHCG6 positive) was transfected with a o vector expressing SP+CRM (a non-functional diphtheria toxin) or vector alone C-l (poDNA3.1) and grown in the presence of neomycin. Four neomycin resistant colonies were individually collected after 14 days and expanded in the continued presence of neomycin. Total mRNA was isolated from each clone and analyzed by RT-PCR using primers PHCG-F and DT-3R. This yielded the predicted 196 bp transspliced product in three out of the four selected clones (Figure 7A, lanes 2, 3 and 4).

The amplified product from clone #2 was directly sequenced, confirming that PTM driven trans-splicing occurred in human cells exactly at the predicted splice sites of endogenously expressed fHCG6 target exon 1 and the first nucleotide of DT-A (Figure 7B).

6.2.8. TRANS-SPLICING IN AN IN VIVO MODEL To demonstrate the mechanism of trans-splicing in vivo, the following experiment was conducted in athymic (nude) mice. Tumors were established by injecting 10 7 H1299 cells into the dorsal flank subcutaneous space. On day 14, PTM expression plasmids were injected into tumors. Most tumors were then subjected to electroporation to facilitate plasmid delivery (see Table 2, below). After 48 hrs, tumors were removed, poly-A mnRNA was isolated and amplified by RT-PCR Transsplicing was detected in 8 out of 19 PTM treated tumors. Two samples produced the predicted trans-spliced product (466 bp) from mRNA after one round of RT-PCLR Six additional tumors were subsequently positive for trans-splicing by a second PCR amplification using a nested set of primers that produced the predicted 196 bp product (Table Each positive sample was sequenced, demonstrating that PHCG6 exon 1 COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:54 FROM- T-966 P058/099 F-541 51 00 0 0 Swas precisely trans-spliced to the coding sequence of DT-A (wild type or CRM OO mutant) at the predicted splice sites. Six of the positive samples were from treatment groups that received cotransfected plasmids, pcPTM+CRM and pcHCG6, which increased the concentration of target pre-mRNA, This was done to enhance the probability of detecting trans-spliced events. The other two positive tumors were N from a group that received only poPTM+Sp (wild type DT-A). These tumors were

O

Snot transfected with OHCG6 expression plasmid, demonstrating once again, as in the C' tissue culture model described in Section 6.2.7, that trans-splicing occurred between Sthe PTM and endogenous PHCG6 pre-mRNA produced by tumor cells.

COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 28-02-'08 a4:54 FROM- T-966 P059/099 F-541 Tabl 2. ns-plicing in tumor; in nudc nice- MnUS Plasnid Left Rin Blcbaporatiou RT-PCR NaitedPCR* Nuclctidc Stquunce 11E1 PCWMrt El-I B1-2 PCMV-por 31-3 31-4 a]ooovI/ 133 PCEj M 3-1 B32 1000CM 83-3 B34 'IO(OOVI S4 pp j B4-1 34-2 ISOVcm B4-3 B4-4 PcSp4CRMI B5-1 BS-2 IOOOV/cm ATOITCCAGJGGCTOAT4R iC(SEQ ID NOM;53) 85-3 H5-4 1OOOVIcm A AT'r rWCCAGjGOGCCGA TfA ID NO,53) 36 PcSp+CRM/ B6-1 6-2 5 B6-3 B64 2SV/vr ATGrCCAGIGGCQTGOrATGA (SEQ ID NO:53) 87 PM4Sp B7.I 410OV- 1E Pc PTM+Sp Es-I bsov/Qwn ATGTCCAGJ3 GCOCTGATQA (SEQ ID N:53) 1B9 PCPTh-Sp B9-1 ATGnOCAGlGOCGTTATGRA ID NO;53) 6 pulses of 9 9 s sets of 3 pulses administered orthogonally b: S pulses of lOis sets of 4 pulses administered orthogonally C: S pulses of 50ms sets of 4 pulses administered orthogonally positive for RT-PCR trans-spliced produce did not receive electroporation 7. EXAMPLE: lacZ TRANS-SPLICING MODEL In order to demonstrate and evaluate the generality of the mechanism of spliceosome mediated targeted trans-splicing between a specific pre-iRNA target and a PTM, a simple model system based on expression of enzyme P-galavtosidas was developed The following section describes results demonstrating successful sp]ioesome mediated targeted trans-splicing between a specific target and a PTM.

COMS ID No: ARCS-i79660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-' 08 14:54 FROM- T96 P6/9 -4 T-966 P060/099 F-541 53 00 7.1. MATEIAL AND METHODS 00 7.1.1. PRMERSEQUENCES The following primers were used for testing the lacZ model system:- Lan-IF C 5 GCATQAATTCGGTACCATGGGCT(0GTWCTCATCATCATC Lac-IR

CTGAOGATCOTCTTACCTGTAAACGCCCATACTGAC

o~3 3Lic-IF GCATGG9TAACCCrGCAGGiGCGG=rCGTCrGGGACTGG 3' Lao-1.R CTGAAAGCTTOT1-AACTTArrATTTTTGACACCAGACC 31 Lao-.Stop OCATG3GTAA.CCCTGCAGci4CGGCTTCOTCTAATAATGOGACTGGGT

G

Is HCG-1n1F GCATOATCCTCCGGAGGGCCCCIN3O3GCACCTTCCAC HCG-TnIR aGACTGCAOGGTAACCGGACAAGGACACTGCTJCACC HCG-.Ex2F.

GC-ATGGTAACCCTGCAGGGGCTCrGCTGrrCiCTO HiCG-Ex2R aGAAAGCTTGEAACCACTCACCATGGTGGGGCAG Lao-flU (Biotin): 7-GGCrTCGCTACCTGGjAGAGAC Lac-TR2 GCTGGATGCGGCGTGCGGTCG HCQ-R2: CGGCACCGTGGCCGAAGTGG 7.1.2. CONS'rMUCTXON OIF THE lacZ PRE-D2RNA TARGET

MOLECULE

The IavZ target 1 pre-znRNA (pc3. I lacTi) was constructed by cloning of the following tee PCR products: the 5 fragment of lacZ; followed by (fi) PHCG6 intron 1; (iii) and the 3' fragment of laoZ. The 5' and 3'fragment of the lacZ COMS ID No: ARCS-1 79660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:54 FROM- T-966 P061/099 F-541 54 00 0 CD gene were PCR amplified fiom template poDNA3.1/His/lacZ (nvitrogen,San Diego, CA) using the following primers: 5' Lac-lF and 5'Lac-lR (for 5' fragment), and 3'Lac-IF and 3' Lac-1R (for 3' fragment). The amplified lacZ 5' fragment is 1788 bp long which includes the initiation codon, and the amplified 3' fragment is 1385 bp 5 long and has the natural 5' and 3' splice sites in addition to a branch point, polypyrimidine tract and pHCG6 intron 1. The pHCG6 intron 1 was PCR amplified using the following primers: HCG-InlF and HCG-InlR.

c The lacZ target 2 is an identical version oflacZ target I except it Scontains two stop codons (TAA TAA) in frame four codons after the 3'splice site.

C 10 This was created by PCR amplification of the 3' fragment (lacZ) using the following primers: 3' Lar-Stop and 3' Lac 1R and replacing the functional 3' fragment in lacZ target 1.

7.1.3. CONSTRUCTION OF pc3.1 PTM1 and pc3.1 PTM2 The pre-tras-splicing molecule, pc3.1 PTM1 was created by digesting pPTM +Sp with PstX and HindEm and replacing the DNA fragment encoding the DT- A toxin with the a DNA fragment encoding the functional 3' end of lacZ. This fragment was generated by PCR amplification using the following primers: 3' Lac-lF and 3' Lac-IR. For cell culture experiments, an EcoRI and HindII fragment of pc3.1 PTM2 which contains the binding domain to HCG intron 1, a 30 bp spacer, a yeast branch point (TACTAAC), and strong polypyrimidine tract followed by the lacZ cloned was cloned into pcDNA3.1.

The pre-trans-splicing molecule, pc3.1 PTM2 was created by digesting pPTM +Sp with PstI and HirndlI and replacing the DNA fragment encoding the DT- A toxin with the pHCG6 exon 2. pHCG6 exon 2 was generated by PCR amplification using the following primers: HCG-Ex2F and HCG-Ex2R. For cell culture experiments, an EcoRI and HindHI fragment of pc3.1 PTM2 which contains the binding domain to HCG intron 1, a 30 bp spacer, a yeast branch point (TACTAAC), and strong polypyrimidine tract followed by the pHCG6 exon 2 cloned was used.

COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:54 FROM- T-966 P062/099 F-541 00 0 0 C) 7.1.4. CO-TRANSFECTION OF THE acZ SPLICE TARGET SPRE-mRNA AND PTMS INTO 293T CELLS 00 0 Human embryonic kidney cells (293T) were grown in DMEM medium supplemented with 10% PBS at 37C in a 5% COz. Cells were co-transfected with 5 pc3.1 LacT1 and pc3.1 PTM2, or pc3.1 LacT2 and pc3.1 PTMI, using Lipofectamine Plus (Life Technologies,Gaithersburg, MD) according to the manufacturer's

\V

instructions. 24 hours post-transfection, the cells were harvested; total RNA was isolated and RT-PCR was performed using specific primers for the target and PTM molecules. p-galactosidase activity was also monitored by staining the cells using a C 10 p-gal staining kit (Invitrogen, San Diego. CA).

7.2. RESULTS 7.2.1. THE lacZ SPLICE TARGET CIS-SPLICES EFFICIENTLY TO PRODUCE FUNCTIONAL P-GALACTOSIDASE To test the ability of the splice target pre-mRNA to cis-splice efficiently, pc3.1 lacT1 was transfected into 293 T cells using Lipfectamine Plus reagent (Life Technologies,Gaithersburg, MD) followed by RT-PCR analysis of total RNA. Sequence analysis of the cia-spliced RT-PCR product indicated that splicing was accurate and occurred exactly at the predicted splice sites (Fig. 12B). In addition, accurate cis-splicing of the target pre-mRNA molecule results in formation of a mRNA capable of encoding active p-galactosidase which catalyzes the hydrolysis of p-galactosidase, i. X-gal, producing a blue color that can be visualized under a microscope. Accurate cis-splicing of the target pre-mRNA was further confirmed by successfully detecting -galactosidase enzyme activity.

Repair of defective lacZ target 2 pre-mRNA by trans-splicing of the functional 3' lacZ fragment (PTM1) was measured by staining for p-galactosidase enzyme activity. For this purpose, 293T cells were co-transfected with lacZ target 2 pre-mRNA (containing a defective 3' fragment) and PTM1 (contain normal 3' lacZ sequence). 48 hours post-transfection cells were assayed for 0-galactosidase enzyme activity. Efficient trans-splicing of PTM1 into the lacZ target 2 pre-mRNA will COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:54 FROM- T-966 P063/099 F-541 56 00 0 0 result in the production of fmotional p-galactosidase activity. As demonstrated in 00 Figure 11B-E, trans-splicing of PTM 1 into lacZ target 2 results in restoration of Pgalactosidase enzyme activity up to 5% to 10% compared to control.

7.2.2. TARGETED TRANS-SPLICING BETWEEN n 5 THE lacZ TARGET PRE-mRNA and PTM2 To assay for trans-splicing, lacZ target pre-mRNA and PTM2 were C transfected into 293 T cells. Following transfection, total RNA was analyzed using 0RT-PCR. The following primers were used in the PCR reactions: lacZ-TR1 (lacZ Ci exon specific) and HCGR2 (pHCGR exon 2 specific). The RT PCR reaction prduced the expected 195 bp rans-spliced product Fig. 11, lanes 2 and 3) demonstrating efficient trans-splicing between the lacZ target pre-niRNA and PTM 2.

Lane I represents the control, which does not contain PTM 2.

The efficiency of the trans-splicing was also measured by staining for p-galaotosidase enzyme activity. To assay for tans-splicing, 293T cells were cotransfected with lacZ target pre-nRNA and PTM 2. 24 hours post-transfection, cells were assayed for p-galactosidase activity. If there is efficient trans-splicing between the target pri-mRNA and the PTM, a chimeric mRNA is produced consisting of the fragment of the iacZ target pre-mRNA and pHCG6 exon 2 is formed which is incapable of coding for an active p-galactosidase. Results from the co-transfection experiments demonstrated that trans-splicing of PTM2 into lacZ target 1 resulted in the reduction of A-galactosidase activity by compared to the control.

To further confirm that trans-splicing between the lacZ target premRNA and PTM2 is accurate, RT-PCR was performed using 5' biotinylated lacZ- TR1 and non-biotinylated HCGR2 primers. Single stranded DNA was isolated and sequenced directly using HCGR2 primer (HCG exon 2 specific primer). As evidenced by the sequence of the splice junction, trans-splicing occurred exactly as predicted between the splice sites (Fig. 12A and 12B), confirming that a conventional pre-mRNA can be invaded by an engineered PTM during splicing, and moreover, that this reaction is precise.

COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:55 FROM- T-966 P064/099 F-541 57 00 0 0 8. EXAMPLE: CORRECTION OF THE CYSTIC FIBROSIS STRANSMEMBRANE REGULATOR GENE 00 Cystic fibrosis (CF) is one of the most common genetic diseases in the world The gene associated with CF has been isolated and its protein product deduced (Kerem, B.S. et al., 1989, Science 245:1073-1080; Riordan et al., 1989. Science 245:1066-1073;Rommas, et al., 1989, Science 245:1059-1065). The protein product of the CF associated gene is referred to as the cystic fibrosis transmembrane conductance regulator (CFTR). The most common disease-causing 0 mutation which accounts for -70% of all mutant alleles is a deletion of three C 10 nucleotides in exon 10 that encode for a phenylalanine at position 508 (AF508). The following section describes the successful repair of the cystic fibrosis gene using spliceosome mediated trans-splicing and demonstrates the feasibility of repairing CFTR in a model system.

8.1 MATERIALS AND METHODS 8.1.1. PRE-TRANS-SPLICING MOLECULE The CFTR pre-trans-splicing molecule (PTM) consists of a 23 nucleotide binding domain complimentary to CFTR intron 9 end, -13 to a nucleotide spacer region (to allow efficient binding ofspliceosomal components), branch point (BP) sequence, polypyrimidine tract (PPT) and an AG dinucleotide at the 3' splice site immediately upstream of the sequence encoding CFTR exon 10 (wild typo sequence containing F508). This initial PTM was designed for maximal activity in order to demonstrate trans-splicing; therefore the PTM included a UACUAAC yeast consensus BP sequence and an extensive PPT. An 18 nucleotide HIS tag (6 histamine codons) was included after wild type exon 10 coding sequence to allow specific amplification and isolation of the trans-spliced products and not the endogenous CFTR. The oligonucleotides used to generate the two fragments included unique restriction sites. (Apal and Pstl, and PstI and NotI, respectively) to facilitate directed cloning of amplified DNA into the mammalian expression vector pcDNA3.1.

COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:55 FROMZ- T-966 P065/099 -541 00 00 The CFTR mini-gen tageoiwa n in Figure 13 and consists Of CFTR exon 9 the- fimotionaql 5' and 3' regions of intron 9 (260 and -265 nurcotidMs tran ac edrespeciel) oxn10( ;SB and the 5'reion of introll 10 (96 nucleotides). in addition, as depicted in Figur 16, a mnini-tre oecmnli IND CFIR exofls 1-9 and 10-24 can be used to test thle use of spliceouollt mediated tratssplicing for Gorrocf oftecyt.fibrosis mutation Fgre 17, shows a double o spacing pINI that mjay also be 'used for correctiOU Of the cystic, fibrosis mutation. As ci ~~~shown, the double splicing PTM1 contains CFrR _Ditot9 peabac on a polypynimidine tract, a, 3' splice site, CFTR exon 10) a Spacer, a branch point, a poiypyrlimidint tract, a 5' splice site and CFTR RD exon 49.1.3.

The following oligonucleotides were used to create CFrR PTM: Forward gF ACCT GOCCC ACC CAT TAKT TAG G3TC A'IT AT CCGCGG Afl ATA Ape, site. Iiflon 9 CFTVR, -12 to 34.

P~eyese Mf4 ACCT gggAGTGACC GIG CAG GALAIA AAA OAK Eonward

CES

ACCT CTGCAG ACT TCA CITOTA ATG ATOAT Patt Exon 10 CT,tl1 to +24 A C GC3C CTA ALTO ATO ATO ATO ALTO ATO Cf C TIC TAG ITO0 OA T Not 1. Stop PolyhistanflC tag 'Exon 10 CFTR, +15 to +132 The following nucleotides were used to create the CVUR TARGET pre-mRNA miii gee (Exo0n 9 iini-Intron 9 Thcon 10*+5- end Intron COMS ID No: ARCS-1 79660 Received by P Australia: Time 15:01 Date 2008-02-18 18-02-'03 14:55 FROM-T-6P6509-41 T-966 P066/099 F-541 59 00 00 F dC1 GACaT CTCGAG OGGA Tfl7 (360 OAK TA Ir oPLO Xhol Exou 9 CRm, 1ito 21.

Ci ~~CFGACCP rCOr7CCGC TAC AcxT OTT (ATTGTC 0Not!. Inton CTGACr (3COOCCGC CCA iCr ATC TC3A AEC

AOT

Not!- introit 9 3' end.

ReoeISO Cr21 OA(XT CTTAAOG TAG ACT PAC CGA ITM AAT jSO Afim The following iionttdfweeused for detection of products: CTA ATO ALTO ATO3 ATO ALTO

ATO

Stop. polyhiime tag biotin label).

ROveSe i-is2 CGC OTA ALTO ATOy ATO ATGY

ATO

3' UT Stop. PolyhistidinS tag biot label).

CM CIT MOT ACr CCa GrC amG Exon 9 CFTR- Forward CE18 GACCE CTCGAG GOA YTi7 060 OAK 'TA WEr GAG xhot- Bxon 9 CFTEL ALAC TAO AAO ciCA CAG TCG AG U a ottre Pc3. ,Ft mesquOC (present in PTM 3' iTbtnttre) COMS ID No: ARCS-i179660 Received by P Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:55 FROM- T-966 P067/099 F-541 00 0 C) 8.2. RESULTS 00 The PTM and target pre-mRNA were co-transfected in 293 embryonic kidney cells using lipofectamine (Life Technologies,Gaithersburg, IMD). Cells were harvested 24 i post transfection and RNA was isolated. Using PTM and targetspecific primers in RT-PCR reactions, a trans-spliced product was detected in which mutant cxon 10 of the target pre-mRNA was replaced by the wild type exon 10 of the ci PTM (Figure 14). Sequence analysis of the trans-spliced product confirmed the Srestoration of the three nucleotide deletion and that splicing was accurate, occurring at o the predicted splice sites (Figure 15), demonstrating for the first time RNA repair of the cystic fibrosis gene, CTR (Mansfield et al., 2000, Gene Therapy 7:1885-1895).

9. EXAMPLE:

QOUBL-ANS-SPLICING

The following example demonstrates accurate replacement of an internal exon by a double-trans-spliing between a target pre-mRNA and a PTM RNA containing both 3' and 5' splice sites leading to production of full length functionally active protein.

As described herein, any pre-mRNA can be reprogrammed by providing a trans-reactive RNAmolecule containing either a 3'-splice site, a site or both. The following example describes successful targeting and replacement of a single internal exon utilizing pre-trans-splicing molecules (PTMs) containing both the 5' and 3' splice sites. Such PTMs can promote two trans-splicing reactions with the intended target gene mediated by the splicesome(s). To test this mechanism, a splicing lacZ model target gene consisting of lacZ 5' "exon" CFTR mini-intron 9 CFTR exon 10 (AF508) CFTR mini-intron 10 followed by lacZ 3' "exon" was created. In this target transcript, a 124 bp central portion of the P-galactosidase

ORF

was substituted by exon 10 (AF508) of CFTR thus it produces non-functional protein.

A PTM consisting of the missing 124 bp lacZ "mini-exon" and a 5' and 3' transsplicing domain containing binding domains (BDs) complementary to the target introns and exons was created. Transfection of HEK 293T cells with either target alone or PTM alone showed no detectable levels of P-gal activity. In contrast, 293T cells transfected with target plus PTM produced substantial lovels of p-gal activity COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:55 FROM- T-966 P068/099 F-541 61 00 0 0 ci d) indicating the restoration of protein function. The accuracy of trans-splicing between L the target and PTM was confrmed by sequencing the appropriate RT-PCR product, 00 which revealed the predicted internal exon substitution. The feasibility of this approach in a disease model was tested by replacing the CFTR AF508 exon 10 with normal exon 10 containing F508 in cystic fibrosis. These results. demonstrate that a N trans-splicing technology can be easily adapted to correct many of the genetic defects Swhether they are associated with the 5' exon or 3' exon or any internal exon of the c-i gene.

O Figure 18 is a schematic of a model laoZ target consisting oflacZ CN 10 oxon CFTRmini-intron 9 CFTR exon 10 (delta 508) CFTRmin-intron followed by the lacZ 3' exon. In this target, a 124 bp central portion of the lacZ gene is substituted with CFTR exon 10 which has a mutation at position 508 (delta 508).

The pre-mnRNA target undergoes normal cis-splicing to produce an mRNA consisting oflacZ 5' exon CFTR exon 10 (delta 508) followed by the lacZ 3' exon. Because of the disruption in p-galactosidase ORF it produces truncated proteins which are nonfunctional.

To restore p-gal function by double-trans-splicing, three PTMs were created consisting of the missing 124 bp lacZ "mini-exon" and a 5' and 3' transsplicing domain containing binding domains complementary to the target introns and exons as shown in Figure 19. These PTMs have an 120 bp 3' binding domain (complementary to intron 9) from PTM24 (see below) used in 3' exon replacement, spacer sequence, yeast branch point, polypyrimidine tract, 3' acceptor AG dinucleotide, lacZ "mini-exon", 5' splice site, spacer sequence followed by the binding domain. These PTMs differ only in their 5' binding domain sequences.

DSPTM5 has a 27 bp BD which is complementary to intron 10 and blocks just the splice site of the target. DSPTM6 has 120 bp 5' BD and covers both 5' and 3' splice sites of the target, while, DSPTM7 has 260 bp BD which masks both the splice sites and and also covers the entire exon of the target.

A schematic representation of a double-trans-splicing reaction showing the binding of DSPTM7 with DSCFTI.6 target pre-mRNA is shown in Figure 20. 3' BD: 120 bp binding domain complementary to mini-intron 9; 5' BD COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'03 14:55 FROM- T-966 P069/099 F-541 62 00 (260) bp); second binding domain complementary to mini-intron 10 and exon 10. s: 00 splice sites; BP: branch point and FPT: polypyrirnidie tract The important structural elements of DSPTM7 (Figure 21) are as follows: a"GAnTCACTnGCTCCA-ATTATCATCCTAGCAGAGTGTATATCflA TTGTAAAGACTATTAACTGTCAAnCATTAAnATACTCT ci GTTrrCATACTCTGCTATGCAC 8 Snace sequence-s bso): AACATTATTATAACGTTGCTCGAA Branch poit pyiidn ratad cetor solice se: 3's BP Kpn 1 PPT BEcoRV .1 IrZ nmini-cxan TACTAAC TGGTACC TGTTCTrrrTTTT GATATC CTGCAC Mlane and 5' donor site and 2 nd sacer sequence: .iatmiiw.I TGA ACrj TAAGT GTTATCACCGATATTGTCTAACCArCGaCC

GATACGCTAAGATCOAGCGG

5' D (60 P): TCAAAAAGTTTCAGATAArrFTTACCrT=T TGAATTCATGC=rTATACCrCTATaTATArTC ATCATTG6CAAACACCAATGATTTCTIATGGTGCC TGGCATAATCCTGGAAACTGATCACATGAAfl CTTCCACTGTGCTAAAAAAACCTCmTG~AnTCTCCA TTTCTCCcATAATCAATnCAACATGAACG TAAAACCCATCArArrAACTCAnTATCATCACGC To determine whether t restoration of p-gal function is RNA tra tasplicing mediated, the mutants are depicted in Figue 22. DSPTh{8 is a 3' -splice mutant in wich the 3' splice elements such asHBP, polypyniidifle trat and the 3' acceptor AG dinucteotides were deleted and replaced with random sequences. This PTM still has 3' and 5' binding domains and the functional 5' splice site. PTM29 lacks *the bindig domain 5' ss but still has the 3' binding domain 3' splice site, while COMS ID No: ARCS-1 79660 Received by IP Australia: Time (I-tm) 15:01 Date 2008-02-18 18-02-'08 14:55 FROM- -966 P07/99 F-541 T-966 PO70/99 F-541 63 00 0 0 (1 PTNO lacks the 1n binding domain 3' splice site but has the functional 5' splice site 00 and 2Va binding domain.

To examine the doube-trans-splicigU mediated restoration of -gal function, 293T cells were either transfected with 2 l.g of target or PTM alone or co-transfected with 2 Ag of target 1.5 Ag ofPTM using Lipofectauine Plus reagent.

48 hrs. after transfection, total RNA was isolated and analyzed by RT-PCR using K IF and Lac-6R primers. These primers amplifyboth cis- and trans-spliced products in a single reaction which were identified based on the size, The cis-spliced product O is 295 bp in size while the tran-spliced product is 230 bp in size. To confirm that trans-splicing between DSPTM7 and DSCFT1.6 pre-nmRNA is precise, RT-PCR amplified products were excised, reamplified using KI -2F and Lac-6R primers and sequenced directly using Kl-2F or Lac-6R primers. As shown in Figure 23 transsplicing occurred exactly at the predicted splice sites, confining the precise intermal exon substitution by two trans-splicing events.

The repair of defective lacZ pre-mRNA by double trans-splicing events and subsequent production of full-length p-gal protein was investigated in cotransfection assays. 293T cells were co-transfected with DSCFT1.6 target and DSPTMI expression plasmids, as well as withDSCT1.6 target or DSPTM7 alone as controls. Western blot analysis of total cell lysates using polyclonal anti-3galactosidase antiserum specifically recognized a 120 kDa protein only in cells cotransfected with DSCFTL.6 target DSPTM7 plasmids (Fig. 24, lanes 3 and 4) but not in cells transfected with either DSCFT1.6 target (Lane 1) or DSPTM7 plasaid alone (Lane Similarly, no full-length protein was detected in cells co-transfected with DSCFT1.6 target 3' splice mutant (Lane 5 and 6) or PTM29 or 30 in which either 3' trans-splicing domain or 5' trans-splicing domains has been deleted (Lane 7).

In addition, the 120 kDa protein band co-migrated with the full-length functional gal produced using lacZ-TI plasmid (positive control, data not shown). These results not only conlrmed the production of full-length protein by double-trans-splicing between the target and PTM but also demonstrated that both the 3' splice site and splice sites are essential for this process.

COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 1-02-'08 14:56 FROM- T-966 P071/099 F-541 64 00 To deter3MiOwertefll- 1lenflgt protein produce ydobe oo liil bewen th are MeRNA ad DSPTI RNIA is funct'gindlY atsi 29 celswer co t protd with PSOFi .6 targeted +4 one of the double t ls p c i n g 7 -9 3 Tv l s w er6 c -r p e si f l p a s m d o r tn n e fe te d w ith D S C F T L 6 ta r g e t O r DSPM1 aone.Total cell extracis were prepared end assayed for P3-al atvt sn NPGM asao(nioe- *3glatvt nexrcSpeae rm cells tranafected WItith,.DTSCFTt.()targ--orDSPTM 7 alnWIamotdnlOlO 0 bakgrflfd leelsdetctedin ocktransfectOl (gig 25). In contrast 293T cells cotranfvetd WtSFTI 6 target and DsPTMi7 produced 21 fold higher le-vels of p~p activity ovrtebcgon Fg Th,5,ts coufinmedthe accurate d~3etas~pjcig between the target Pre-mPNA and PTM RNA anad productioni of the full-length functional Protein. fPSlatvt y obefasslcn TO confirm that restou of bot 3, actid by sp~lic~estesdflftli reactiO~h is absolutely depended on the prese fbt 'sA5 sc sites ft iS L we construce several mutantt pSpTMS, is identical 0 t M7ecp the Y sice elements (branch point, polyp3'fl idfl~c tract and the 3' atcePtU AGe incltin s) wer d ltd and substit t d with ra do mi Seque c e (see Fig. 22 foir details); (b)flTM2 9 lacks 5spiestaswlashe'bndndo andtha 3'bnigdomain and 3' splice site, and PTM3O lacks 3' bindingS dInnltnd 3' splice site but has the 5' splice site and or bidn doa. P-gal actviyne ewtacs prepaed fom clls ranfeted with either pSCPTI .6 targetorD TM aonws almost identical to the baeltrlid leve s detected in col tasetllsi (wfig.26) similarly, no0 signfitlOt increase in P3-gal activity w as ectin ell iDSCfeT 1d wIt either P)SPTMB alone splice sit muat ho o-ancsf Otraut 0 fSC tl.

taget oneof the above mutant PTMB.5 O~n the other hncls 0 ~rtfodwt target and DSOn"w t functiofll 3 and 5-'splie sites Produced substantial levels of il-gal activity over the SckgroU p6.Tee eut conufirmed fth requlirelnent of both splice sites in thle doublO.4Phtm PTMI and also eliminated, the possibilithtat restoration Of 1-gal activity wsdue t comUplernentatlon between the truncated Proteins (Fig. 26).

COMS ID No: ARCS-179660 Received by P Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:56 FROM- T-966 P072/099 F-541 0 0 C) Different concentrations of the target and PTM were co-transfected OO and analyzed for p-gal activity restoration. As expected, 293T cells co-transfected with DSCFT1.6 target DSPTM7 showed substantial levels of p-gal activity fold) over the controls. Increasing the concentrations of the PTM by 2 and 3 fold did increase the level of 1-gal activity, but not significantly (Fig. 27), These results u further confirmed the double-trans-splicing mediated restoration of p-gal enzyme function.

Cl The specificity of double-trans-splicing reaction was examined by 0 0 constructing a non-specific target (DSHCGT1.1) which is similar to that of specific target (DSCFT1.6) but has PHCG intron 1 pHCG exon 2 and pHCG intron 2 instead of CFTR mini-intron 9 CFTR exon 10 (delta 508) and CFTR mini-intron 10 (Fig.

28). RT-PCR analysis of the total RNA isolated from cells transfcted with either DSHCGT1.1 (non-specific target) alone or in combination DSPTM7 (targeted to DSCFTI.6 target) failed to produce the expected 314 bp double-trans-spliced product.

On the other hand, RT-PCR analysis of the total RNA prepared from cells cotransfected with specific target PTM produced the expected 314 pb product. This was further confirmed by P-gal activity assay of the total cellular extract. The level 3gal activity detected in cells transfected with non-specific target alone or in combination with DSPTM7 targeted to DSCFT1.6 target was almost identical to the background level. In contrast substantial levels of p-gal activity was detected in cells co-transfected with specific target (DSCFT1.6) DSPTM7 (Fig. 27). These results confirmed that the double-trans-splicing is highly specific.

The repair model in Fig. 30 shows a portion of a target CFTR premRNA consisting of exons 1-9, mini-intron 9, exon 10 containing the delta 508 mutation, mini-intron 10 and exons 11-24 (Fig, 30). The PTM shown in the figure consists of exon 10 coding sequences (containing codon 508) and two trans-splicing domains each with its own splicing elements (acceptor and donor sites, branchpoint and pyrimidine tract) and a binding domain complementary to intron 9 splice site, part of exon 10 and 3' ends) and intron 10 5' splice site (Fig. 31 (DS-CF1)). Exon 10 of the PTM also has modified codon usage throughout to reduce antisense effects between exon 10 of the PTM and it's own binding domains and for PTMs that have COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:56 FROM- T-966 P073/099 F-541 66 00 0 0 binding domains which are complementary to exon sequences (Fig. 31). A double- 00 trans-spHcing event between the PTM and target should produce a repaired fulllength mRN- Fig. 32 shows the sequence of a single PCR product showing target exan 9 correctly spliced to ?TM 20 exon 10 (with modified codons) (upper panel), O codon 508 in exon 10 of the PTM (middle panel) and PTMn exon 10 correctly spliced cod'On 508 i n =on w a generated by C to target exon 11 (lower panel). The sequence of a epaid targtwaS RT-PCR followed by PCR EXAMPLE: TAS-SPLICN G REPAIR OF THE CYSTIC pIBROSIS (ENSI USING A PTv THAT CAN PERFORM 5' EXON RPLACEENT The key advantage of using 5' exon replacement for gene repair are it penits replacement of the 5' portion of a gene the construct requires less sequence and space than a full-length gene construt, PTMs can be produced that lack a polyA signal which should prevent PTM translation, and the 5' end can be modified to increase translation.

10.1 MJTSAb

METHDS

10.1.1 PIASM CONMUCT The CFTR coding sequences (exons1-10) for PTM30 were generated by PCR using a partial DNA plasmid template (61160; American Type Culture Collection. Manassas, VA). The trans-splicing domain (TSD) [including the binding domain, spacer sequence, polypyimidiu tract (PPT), branchpoint and 3' splice site] was generated from a PCR product (using an existing plasmid template) and by annealing oligonucleotides. The difetent fragments (the TSD and coding sequences) were then cloned into pcDNA3.1(-) using appropriate restiction sites.

Oligodeoxynocleotide primers were procured from Sigma Genosys (The Woodlands, TX). All PCR products were generated with either REDTaq (Sigma, St. Louis, MO), or cloned Pfiu (Stratagene, La Jolla, CA) DNA Polymafse. PCR primers for COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:56 FROM- T-966 P074/099 F-541 67 00 0 0 amplification contained restriction sites for directed cloning. PCR products were 00 digested with the appropriate restriction enzymes and cloned into the mammalian expression plasmid pc3.1IDNA(-) (Invitrogen Carlsbad,

CA).

10.2 CULTURE AND TRANSECTIONS V 5 Constructs were cotransfected in human embryonic kidney (HEK) 293T or 293 cells (1.25 x 106 cells per 60 mm poly-d-lysine coated dish) using SLipofectamuinePlus (Life Technologies, Gaithersburg, MD) and the cells were O harvested 48 h after the start of transfectionL Total RNA was isolated as described in the manufacturers instructions (Epicenter Technologics, Inc.). I-EK 293T cells were grown in Dulbecco's Modified Eagle's Medium (Life Teclmologies) supplemented with I0o v/v fetal bovine seum (Hyclone. Inc., Logan, UT). All cells were kept in a humidified incubator at 37 OC and 5% COz.

10.1.3 REVERSE

TRANSCRIPTION-POLYMEASE

CHAN REACTION

(TR-PCR)

RT-PCR was performed using an EZ-RT-PCR kit (Perkin-Ehnler, Foster, CA). Each reaction contained 0.03 to 1.0 Mg of total RNA and 80 ng of a and 3' specific primer in a 40 jl reaction volume. RT-PCR products were electrophoresed on 2% Seaken agarose gels. The PTM- and target-specific oligonucleotides used to generate trans-spliced products are 5'-CGCTGGAAAAACGAGCTTGTTG-3' (primer CF93) and 3 (primer CF111), respectively. The PTMand target-specific oligonueleontides used to generate cis-spliced products were CF1 and CF93. The sequence of oligonucleotide CF1 is ATGAT ATGG-3'.

The repair model in Fig. 33 shows a portion of a target CFTR premRNA consisting of exons 1-9, mini-intron 9, exon 10 containing the delta 508 mutation, inini-intron 10 and exons 11-24 (Fig. 33). The PTM shown in the figure consists of exon 1-10 coding sequences (containing codon 508) and a trans-splicing domain with its own splicing elements (donor site, brancbpoint and pyrimidine tract) and a binding domain. Several PTMa have been constructed with different binding COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:56 FROM- T-966 P075/099 F-541 68 00 0.

0 ci r. domains. Three examples are shown in Figure 34. In Fig. 34A the binding domain is 00 complementary to the splice site of intron 9 and part of exon 10 end; CF-PTM 11).

In Fig. 34B the PTM has an extended binding domain which also covers the 5' end of exon 10 and the 3' splice site ofintron 9 (CF-PTM 20). In the last example (Pig. 34C) It 5 the binding domain is the same as that shown in panel B except the binding domain Va extends the full-length of exon 10 (CF-PTM 30). In the latter case the PTM exon has modified codon usage to reduce antisense effects with it's own binding domain CN (Fig. 34). Further examples of binding domains rxe shown in Figure o Figure 36 shows the sequence of cis- and trans-spliced products. The top panel of Fig. 36A shows target exon 10 with it's three missing nucleotides (CTT), whilst the lower panel shows exon 10 and 11 of the target correctly spliced together.

Figure 36B is a partial sequence of a single PCR product showing the modified codons in exon 10 of the PTM (upper panel), codon 508 in exon 10 of the PTM (middle panel), and PTM exon 10 correctly spliced to target exon 11 (lower panel), indicating that tranz-splicing is accurate. The sequence of the repaired target was generated by RT-PCR followed by PCR.

11. EXAMPLE: P WITH A LONG BINDING DOMAIN, V, ilCH MAY BE DISCONTINUOUS, HAVE INCREASED TRANS-SPLICING EFFICIENCY AND SPECIFICITY 11.1. MATERIALS AND METHODS 11.1.1. CELL CULTURE Human embryonic kidney cells (293 or 293T) were from the University of North Carolina tissue culture facility at Chapel Hill (Chapel Hill, NC).

Cells were maintained at 37 0 C in a humidified incubator with 5% CO 2 in Dulbecco's modified Eagle's medium (Life Technologies, Bethesda, MD) supplemented with v/v fetal bovine serum (Hyclone, Logan, UT). Cells were passaged every 2-3 days using 0.5% trypsin and re-plated at the desired density. Stable cells, expressing an endogenous mutant lacZ pre-mRNA (labZCF9) were maintained in the presence of 0.5 mg/nm G418 (Calbiochem, San Diego, CA).

COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:56 FROM- T-966 P076/099 F-541 69 00 0 0 11.1.2. RECOMBINAT PLASMIDS 00 Targets: pc3.llacZCF9, pc3. llacZCF9m, and pc3.llacZRCGm.

pc3.llacZCF9 encodes for a normal lacZ pre-mRNA was constructed using lacZ coding sequences nucleotides 1-1788 as 5' exon, CFTR mini-intraon 9 followed by f 5 lacZ coding sequences nucleotides 1789-3174 as 3' axon. This is similar to NOpc3.llacZ-T2 construct but vithout stop codons in the lacZ 3' oxon and has CFTR C mini-intron 9 instead of PHCG6 intron 1 (Fig. 37A). CFTR mini-intron 9 was PCR amplified using plasmnid TS as template and primers CFIN-9F o CTAGGATCCCGTTCTrGTT CACT ATTAA) and CFIN-9R CTAGGQGTTACCGAAGTAAAACCATACTTATTAG, restriction sites underlined), digested with BamnH I and BstE II and cloned in place of BHCG6 intron 1 of pc3.llcZ-T2 plasmid. pc3.llaZCF9m expresses a defbctive lacZpre-mRNA and is identical to pc3.11acZCF9 but contains two in-frame non-sense codons in the 3' exon (Fig. 37A). pc3.lacZICGlm is a chimeric target, which includes the lacZ 5' exon followed by intron 1 and exon 2 of 0HCG6. This is similar to po3.llacZCF9m except that it contains exon 2 of 3HCG6 in place of mutant lacZ 3' aexon. PHCG6 exon 2 was PCR amplified using PHCG6 plasmid (accession X00266) as template DNA and primers HCGEx-2F GCATGGTTACCCTaGCAGGGGCTGCTGCTGTTGCTG) and HCGEx-2R CTGAAAGCTTGTTAACCAGCTCACCATGGTGGGGCAG, restriction sites underlined) digested with BRtE II and hind III and cloned in place of the lacZ 3' axon of pc3.llacZCF9nm. Plasmid pcDNA3.1/isB/lacZ (Invitrogen, Carlsbad. CA) was used as DNA template to produce 5' and 3' laZ exons. The lacZ 5' exon is 1788 bp long, has an ATG initiation codon, lacZ 3' exon (without stop codons) is 1385 bp long and has a transcription termination signal at the end of the 3' exon. CFTR mini-intron 9 and |HCG6 intron 1 are 548 bp and 352 bp in size, respectively, and both have and 3' splice signals. Exon 2 of PHCG6 is 162 bp long and has a transcription tennination signal at the end of the exon.

Pre-trans-splicing Molecules (PTMs): PTM-CFl4 is an identical version of pcPTM1 with minor modifications in the rrans-spliting domain (Fig. 37B).

PTM-CF14 is a linear version and contains a 23 bp antisense binding domain (BD) COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:57 FROM- T-966 P077/99 F-541 00 0 TAGGTCATTAT) complementary to CFTR mini-intron 9, 00 18 bp spacer, a canonical branch point sequence (UACUAAC; BP) and an extended polypyrimidine tract (PPT) followed by normal lacZ3' exon. PTM-CF22,

PTM-

CF24, PTM-CF26 and PTM-CF27 are identical to PTM-CF14 except they differ in 5 length ofthe BD (Fig. 37B). sPTM-CF18 has a 32 bp BD, sPTM-CF22 and sPTM- SCF24 contain the same BD as PTM-CF22 and PTM-CF24, respectively. In these PTMs, the binding domains were modified to create intra-molecular stem-loop o structure C(safety) to mask the 3' splice-site of the PTM. Different binding domains were produced by PCR amplification using specific primers (with unique NIe I and Sac 1 sites) and a plasmid containing CFTR mini-intron 9 as template. PCRproducts were digested with Nhe I and Sac II and cloned into a PTM plasmid consisting of spacer sequences, 3' splice elements (BP, PPT and acceptor AG dinucleotide) followed by a normal lacZ 3' exon.

11.1.3. TRANSFECTIO OF PLAS DA INTO 293T LL The day before transfection, I x 106 293T cells were plated on 60 mm plates coated with Poly-D-lysine (Sigma, St. Louis, MO) to enhance the adherence of cells and grown for 24 hr at 37°C. Cells were transfected with expression plasmids using LipofectaminePlus reagent according to standard protocols (Life Technologies, Bethesda, MD). In a typical co-transfection, 2 pg ofpo3.llacZCF9m target and 1.5 pg of PTM expression plasmids were transfected into cells and for controls (target and PTM alone transfections) total DNA concentration was normalized to 3.5 pg with pcDNA3.1 vector.

Forty-eight hours after transfectionthe plates were rinsed with PBS, cells harvested and total RNA or DNA was isolated using MasterPure

RNA/DNA

purification kit (Epicenter Technologies, Madison, WI). Contaminating DNA in the RNA preparation was removed by treating with DNase I, while, contaminating

RNA

in the DNA preparation was removed by digesting with RNase A at 37°C for 30-45 min.

COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:57 FROM- T-966 P078/099 F-541 71 00 0 0 11.1.4. REVERSE TRANSCRIPTION-POLYMERASE CHAIN REACTION (RT-PCR1 00 RT-PCR was performed as suggested by manufacturer using an BZ rTth UNA PCR kit (Peridns-Elmer, Foster City, CA). A typical reaction (50 L) contained 25-500 ng of total RNA, 100 ng of 5' target specific primer (common to cisand trans-spliced products) (Lac-9F, S'PGATCAAATCTGTCATCCTTCC) and 100 ng of3' primer (Lac-3R, 5'-CTGATCCACCCAGTCCATTA, target specific primer for cis-splicing, and Lac-5R 5GACTGATCCACCCAGTCCCAGA,

PTM

o specific primer for trans-splicing), IX reverse transcription buffer (100 mM Tris-HC, pH 8.3, 900 mM KCL with 1 mM MnClz), 200 MM dNTPs and 10 units of rIth DNA polymerase. RT reactions were perfonrmed at 60C for 45 min. followed by 30 sec pre-heating at 94C and 25-35 cycles ofPCR amplification at 94*C for 18 se, annealing and extension at 60"C for 1 min followed by a final extension at 70C for 7 rin. The reaction products were analyzed by agarose gel electrophoresis.

11.1.5. PROTEINPREPARATION AND -GAL ASAY Total cellular protein from celiq transfected with expression plasmids was isolated by freeze thaw method and assayed for p-galactosidase activity using a.

P-gal assay kit (nvitrogen, Carlsbad, CA). Protein concentration was measured by the dye-bin ading assay using Bio-Rad protein assay reagents (BIO-RAD, Hercules,

CA).

11.1.6. WESTERN BLOT About 5-25 £g of total protein was electrophoresed on a 7.5% SDS- PAGE gel and electrobiotted onto PVDF-P membrane (Millipore). After blocking for 1 hr at room temperature (blocking buffer: 5% dry milk and 0.1% Tween-20 in IX PBS), the blot was incubated with a 1:2500 dilution of polyclonal rabbit antigalactosidase antibody for hr at room temperature (Research Diagnostics Inc. NJ), washed 3x with blocking buffer and then incubated with a 1:5000 diluted anti-rabbit HRP conjugated secondary antibody. After incubating at room temperature for 1 hr, COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:57 FROM- T-966 P079/099 F-541 72 00 0 0 it was washed 3x in blocing buffer and developed using ECLPlus Wester blotting Sreagents (Amersham Pharmacia Biotech, Piscataway,

NJ).

11.1.7. ]INSTUm-GA

STAINIIG

t Cells were monitored for the expression of functional p-galactosidase using a P-gal staining kit (Invitrogen, Carlsbad, CA). The percentage of -gal positive cells were deterined by counting stained vs. unstained cells in 5-10 o randomly selected fields.

11.1.8. SELRCTION OF NEOMYCIN RESISTANT CLO ES EXPRESSING AN ENDOGENOUS DEFECTIVE lacZ pf.TR1A

TARGET

On day 1, 1 x 106 293 cells were plated on 60 rm plates and grown for 24 br at 37C. On day 2, the cells were transfected with 2 g of p-3.1 1 oZCF9m using LipofectaminePlus transfection eagent as described above. 48 hr posttransfection, cells were split (1:20 ratio) and grown in media containing 0.

5 mg/m 1 G418. At the end of 2 weeks, neomycin resistant colonies were slected, pooled expanded and maintained constantly in the presence of G418.

11.2. RESUTS A model system was developed that permits facile and versatile analysis of spliccosome mediated RNA ans-splicing in cells. The bacterial lacZ gene was split with a truncated intron 9 from the Cystic Fibrosis Transembrane Conductance Regulator (CFTR) gene (Figure 37A). This split lacZ gene, when introdced into human 293T cells, directed the synthesis of a lacZ pre-RNA that could splice properly. The opea reading frame of the IacZ gene was mutated by insertion of two in-frame nonsense codons near the 5' end of the second exon (Figure 37A). This lacZ gene is referred to as lacZCF9m. In 293T cells, laoZCF9m directs the synthesis of lacZCF9m pre-mRNA, which encodes a truncated p-galactosidase

(P-

gal) protein that does not have enzymatic activity. Cells bearing the lacZCF9m gone are a model system for genetic disorders caused by loss of function mutations.

COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:57 FROM- T-966 P080/99 F-541 73 00 0 0 prei.rans-splicing molecules (PTMs) were designed to traLns-spice 0O awithlacZCF9mpre-mRNA and repair the mutation causedby the two nonsense codons. PTMs were constructed with binding domains spanning 23, 91 and 153 nucleotides which we named T-C 14 PTM-CF4,PTM -22 and PTM-CF24 (Figure 37B). The PTM-CF24 binding domain does not bind 153 contiguous at in the O targeted CFTR gene intron 9, but rather creates a loop of 4 nt in the target in between two regions ofcomplimeatary of 27 and 126 nt (Figure These PTMs were' predicted to repair the deficiency created by lacZCF9n (Figure 37C).

o Semi-quantitativeRT-PCR analysis was used to tests the efficiency of trans-splicing mediated by PTMs with long target binding domains. Repair of lacZCF9m transcripts by trans-splicing was tested in two different ways: ctransfection of PTM and target (lacZCF9m) plasmids or tranasfection of cells that had been modified to express the target as an endogenous pre-mRNA- Co-transfecting plasmids encoding PTMs with the lacZCF9m plasid provided a facile method for screening the former for efficiency. PTM-CFZ2 and PTM-CF24 were approxilmately 3-fold and 10-fold more effiient than PTM-CF14 in a sei-quantitative

RT-PCR

assay suggesting a significant improvement in mRNA repair (Figure 38). Sequencing of the RT-PCR products showed that trans-splicing was accurate, resulting in proper ligation of the exons from the target and the PTM. Moreover, mutation of key ciacting elements in the 3' splice site of the PTMs resulted in an abrogation of transsplicing. In these and all other assays described herein controls were carried out to rule out recombination at the DNA level. Thus, repair of the lacZCF9m transcripts was a result of targeted PNA traln-sphcing.

Transfection of PTM-CF14, -CF22 or -CF24 into 293 cells bearing an endogenous lacZCF9 gene contfnued that the longer target binding domains provided the PTMs with higher efficiency (Figure 38B). It should be noted that similar levels of RT-PCR trans-splicing specific product were obtained after 30 PCR cycles and 35 cycles for PTM-CF24 and P'r-CF14, respectively. The datatherefore suggests that PTMs with long binding domains repaired lacZCF9m transcripts at least an order of magnitde better than previously described PTMs.

COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:57 FROM- T-966 P081/099 F-541 74 00 0 0 ci d0 More than one in ten transcripts of lacZCF9m can be repaired by transsplicing. Quantitative, real-time PCR was used to measure the fraction of lacZCF9m 00 transcripts repaired by PTMs with long binding domains. The co-transfection assay described above was used in these experiments. PTM-CF14, which contains a binding domain of 23 nt, was shown to repair between 1-2 and 1.6% oflacZCF9m RNAs in 293T cells and 2.1% of lacZCF9m RNAs in the H1299 human lung cancer

\O

cells. PTM-CF24, which has a 153 not long binding domain, was significantly more t C efficient, correcting between 12.1 and 15.2% of lacZCF9m RNAs in 293T cells and 0 19.7% in H1299 cells. This in effect resulted in a measurable reduction in the levels Ci 10 of lacZCF9m mRNA. These data also confirmed the remarkable capability of this RT-PCR assay to distinguish between the products of cis-splicing, the lacZCP9m and mRNA, and the products of transs-plicing, repaired lacZCF9m mRNA. This is the first true quantification of the efficacy of trans-splicing mediated mRNA repair at the RNA level. These data confirm the suggestions of the semi-quantitative RT-PCR analysis shown above. Similar experiments were carried out using 293 cells that express an endogenous lacZCF9m pre-mRNA target Consistent with the data shown above, PTM-CF24 was ten times more efficient than PTM-CF14, with the former correcting between 1.3 and 4.1% of endogenous lacZCF9m transcripts. These data confirmed that increasing the length of the PTMs provided a remarkable enhancement in trans-splicing efficiency.

Trans-splicing mediated mRNA repair results in the synthesis of active P-galactosidase. At the cellular level, the ultimate criterion for the success of miRNA repair is the production of an active protein. Using a western assay it was determined that full-length P-gal was produced as a result of trans-splicing. Full-length p-gal was not observed following transfection of 293T cells with plasmids encoding lacZCF9m or PTM-CF24. Co-transfection of both plasmids, however, resulted in robust production of full-length p-gal protein, which was readily detectable using anti-p-gal antiserum (Figure 39). This result complements enzymatic activity data suggests that the latter was not due to a complementation by truncated p-gal proteins.

The Western blot analysis revealed that full-length p-gal protein was made in 293T COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:57 FROM- T-966 P082/099 F-541 00

O

O

cells by trans-splicing and furthermore confrmed that the PTMs with long binding 0 domains wre efficiently splied.

Appropriate repair of P-gal mRNA and synthesis of full-length p-gal protein should lead to the production of active enzyme. Indeed, 293T cells cotransfected with lacZCF9m and PTM-CF24 were shown to have p-gal activity S measured either in situ (Figure 40A) or in extracts (Figure 40B). This activity was shown to depend on the trans-splicing between the target pre-mRNA and the PTM.

CI The quantitative in solution assay further confinned the data presented above: PTMo CF22 and PTM-CF24 were 2.9 and 9.3 fold more efficient respectively than PTM- C 10 CF14. Most impressive, however, were results using 293 cells that harbor lacZCF9m as a stable endogenous gene. When these cells were transfected with PTM-CF14 the levels of 3-gal activity obtained were barely above background. Transfection with PTM-CF24, however, resulted in a considerable level of p-gal activity (Figure This was paralleled by the appearance of full-length p-gal protein. These data S 15 demonstrate a sizeable increase in the efficiency of trans-splicing to repair a mutated pre-mRNA. In fact all prior reports of repair of endogenous RNA in mammalian cells by either group I ribozymes or trans-splicing have been only documented using RT- PCR, an indication of the low level of repair.

PTMs with very long binding domains are highly specific. It was shown that a secondary structure within the binding domain could enhance specificity of PTMs in HeLa nuclear extracts. In order to ascertain the specificity of the transsplicing reactions in vivo a second target gene was prepared, which could serve as reporter ofnon-specific reactions. This gene, which is referred to as lacZHCGln, shares the first exon with lacZCF9m. The intron in lacZHCGlm is intron 1 of the Psubunit of the human chorionic gonadotropin gene 6 (phCG6) and the second exon is exon 2 of the same gene. lacZHCGlm drives the synthesis of a pre-mRNA that is spliced correctly to yield a chimeric raRNA that does not encode a full-length P-gal (see below). PTM-CF14, -CF22 and -CF24 are not targeted to laoZHCGlm premRNA since there is no complementarity between the binding domains in these PTMs and the target gene. Any trans-splicing between these PTMs and laoZHCGlm pre-mRNA is therefore non-specific (Figure 41A).

COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:58 FROM- T-966 P083/099 F-541 76 00 O0

O

1) 293T cells were transfected with PTM-CF14, -CF22 or -CF24 and the Slevel of non-specific trans-splicing was determined by RT-PCR and by in solution 00 P-gal assays. Semi-quantitative RT-PCR suggested that PTM-CF24 was significantly less likely than PTM-CF14 to trans-splice with lacZHCGlm pre-mRNA.

Measurement of 0-gal activity confirmed this; cells co-transfected with lacZHCGlm and PTM-CF24 produced 3.7 fold less p-gal than those co-transfected with lacZHCG1m and PTM-CF14 (Figure 41C). Based on these data it was estimated that SPTM-CF24 is 50 times more likely to trans-splice to its target than to a non-specific target. A "safety" version of PTM-CF24, sPTM-CF24, did not confer further Ci 10 specificity (Figure 41C). Nonetheless, for PTMs with shorter binding domains a "safety" stem involving the binding domain was seen to improve specificity in vivo (Figure 41C). It was concluded from these data that the longer binding domains resulted in PTMs that were not only more efficient but also more specific.

The observation that long binding domains increased the specificity of PTMs suggested that very long binding domains (C200 nt) could further enhance discrimination. Plasmids encoding PTM-CF26 and -CF27, which have binding domains that span 200 nt and 411 nt respectively, were constructed and co-transfected with lacZHCGlm plasmid. Non-specific trans-splicing of these two PTMs was barely detectable with RT-PCR (Figure 41B). As measured by the p-gal assay PTM- CP26 and -CF27 had minimal non-specific trans-splicing activity (Figure 41C). In a specific trans-splicing reaction with lacZCF9m as measured by the solution P-gal assay PTM-CF26 was as active as PTM-CF14 (Figure 41B). It was estimated that PTM-CF26 is 80 times more likely to trans-splice to the specific target (acZCF9m) than to a non-specific target (acZHCGlm). Therefore, inclusion of very long binding domains confers to these PTMs very high specificity.

12. EXAMPLE: CORRECTION OF THE FACTOR Vm GENE USING 3' EXON REPLACEMENT Hemophilia is a bleeding disorder caused by a deficiency in one ofthe blood clotting factors. Hemophilia A, which accounts for about 80 percent of all cases is a deficiency in clotting factor VIII. The following section describes the COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:58 FROM- T-966 P084/099 F-541 77 00 0 0 ci 1) successful repair of the clotting factor VIII gene using spliceosome mediated transsplicing and demonstrates the feasibility of repairing the factor VIMI using gene 00 therapy.

The coding region for mouse factor VIH PTM (exons 16-24) was PCR amplfied from a cDNA plasmid template using primers that included unique restriction sites for directed cloning. All PCR products were generated with cloned Pfu DNA Polymerase (Stratagene, La Jolla, CA). The coding sequence was cloned C^ into pc3.1DNA(-) using EcoRV and Pmel restriction sites. The binding domain (BD) O was created by PCR using genomic DNA as a template. Primers included unique C' 10 restriction sites for directed cloning. The PCR product was cloned into an existing PTM plasmid (PTM-CF24, pc3.1DNA) using NheI and Sacl restriction sites. This plasmid already contained the remaining elements of the TSD including a spacer sequence, polypyrimidine tract (PPT), branchpoint (BP) and 3' acceptor site. The whole of the TSD was then subcloned into the vector (described above) containing the factor VII PTM coding sequences. Finally, bovine growth hormone 3' untranslated sequences from a separate plasmid clone were subcloned into the above PTM using Pmei and BamHI restriction sites.

The whole construct was sequenced and then analyzed by RT-PCR for possible cryptic splicing, and then subcloned into the AAV plasmid pDLZ20-M2 using Xhol and BamHI restriction sites (Chao et al., 2000, Gene Therapy 95:1594- 1599; Flotte and Carter, 1998, Methods Enzymol., 292:717-32). For some viral (and non-viral) delivery systems, the size of the therapeutic is essential. Vital vectors such as adeno-associated virus are preferred because they are a non-pathogenic virus with a broad host range (ii) it induces a low inflammatory response when compared to adenovirus vectors and (iii) it has the ability to infect both dividing and non-dividing cells. However, the packaging capacity of the rAAV is limited to approximately 110% of the size of the wild type genome, or -4.9 kB, thus, leaving little room for large regulatory elements such as promoters and enhancers. The B-domain deleted human factor VIII is close to the packaging size of AAV, thus, trans-splicing offers the possibility of delivering a smaller transgene while permitting the addition of regulatory elements.

COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:58 FROM- T-966 P085/099 F-541 78 00 0 0 1) To eliminate cryptic donor sites in the pre-mRNA upstream of the 00 AhoI PTM cloning site approximately 170 bp of sequence was eliminated from the original AAV construct that includes part of exon 1 and all of the intron I sequence (see Fig. 44C).

The repair model in Fig. 44D shows a simplified model of the mouse C. factor VII pre-nmRNA target (endogenous gene) consisting of exons 1-14, intron 14, exon 15, intron 16, and exon 16-24 containing a neomycin gene insertion. The PTM Ci shown in the figure consists of exon 16-24 coding sequences and a trans-splicing o domain with its own splicing elements (donor site, branchpoint and pyrimidine tract) and a binding domain. Details of the binding domain are shown in Fig. 44A and 44B.

The binding domain is complementary to the splice site of intron 15 and part of exon 16 end).

The key advantages of using 3' exon replacement for gene repair are (i) the construct requires less sequence and space than a full length gene construct, thereby leaving more space for regulatory elements, (ii) SMaRT repair should only occur in those cells that express the target gene, therefore eliminating any potential problems associated with ectopic expression of repaired RNA.

Factor VIII deficient mice were maintained at the animal facilities at the University of North Carolina at Chapel Hill. For plasmid injections each mouse was sedated and placed under a dissecting microscope and a 1 cm vertical midline abdomen incision was made. Approximately 100 micrograms of PTM plasmid DNA in phosphate buffered saline was injected to liver portal vein. Blood was collected from the retro-orbital plexus at intervals of 1, 2, 3 and 20 days after injection and assayed for factor Vm activity using the Coatest assay.

Factor VIII activity in blood samples collected from mice were assayed using a standard test called the Coatest assay. The assay was performed according to manufacturer's instructions (Cbromgenix AB, Milan, Italy). Data indicating repair of factor VIII in factor VII knock out mice is demonstrated in Figure 46.

Hemophilia A defects in humans are broadly split into several categories that include gross DNA rearrangements, single DNA base substitutions, deletions and insertions. It has been determined that a rearrangement of DNA COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:58 FROM- T-966 P086/099 F-541 79 00 0 Sinvolving an inversion and translocation of exons 1-22 (together with introns) away 00 from exons 23-26 is responsible for -40% of all cases of severe hemophilia A. The canine hemophilia A model also has a very similar gross rearrangement. This mutation will be used as the basis for our human and canine factor V IPTM designs.

5 Methods for building the human factor VI PTM will be very similar V to that described above for the mouse PTM except that different coding regions S(exons 23-26) will be amplified from a human cDNA, the binding domain will be C amplified from human genomic sequence templates (whole genomic DNA or a o genomic clone), and a C-terminal FLAG tag will be engineered in the PTM to be used

C

N 10 to detect repaired factor VII protein. The remaining elements of the trans-splicing domain including a spacer sequence, polypyrimidine tract (PPT), branchpoint (BP) and 3' acceptor site will be obtained from an existing plasmid. Where necessary changes will be made to the binding domain sequence to eliminate any cryptic splicing within the PTM. The final PTM will be subcloned into the same mouse AAV plasmid vector, pDLZ20-M2 and virus preparation made from this plasmid. The canine factor Vm PTM will be made in an identical fashion but using canine cDNA and genomic plasmid.

13. EXAMPLE: TARGETED TRANS-SPLICING

OF

PAPILLOMAVIRAL RA The vast majority of cervical cancers are associated with oncogenic hunan papilloma viruses (HPVs) and express viral mRNAs encoding the E6 and E7 oncoproteins. As described below, PTMs targeted against the E6 region of HPV-16 and splice the TM cxon to the 5' end of the E6 ORF using the 5' splice site at the nucleotide 226.

13.1 MATERIALS AND METHODS The target DNA (pl059) was used to test PTM efficiency and contains the entire HPV-16 early region (nt 79-4468) cloned behind the SV40 early promoter and origin of replication. Specificity was assessed using the heterologous expression vector lacZCF9m as target (Puttaraju e al. 2001. Mol Ther 4:105-14). Plagmids were prepared using Quiagen maxi prep kits.

COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:58 FROM- T-966 P087/099 F-541 00 0 Nearly confluent 6 cm plates of 293 cells were transfected with 2 pg 00 target DNA and 2 gg PTM DNA using LipofectAmine 2000 (Life Technologies). At two days post-transfection, cells were washed on the plate with PBS and lysed on the plate using 300 pl lysis buffer. Total cell RNA was prepared using Ambion RNAqueous kit Transfected DNA was removed from the RNA by LiCI precipitation followed by DNAse I treatment using the Amboin DNA-freeV DNAse treatment and removal reagents.

Ci RNA was converted to cDNA using RT from the High Capacity oDNA SArchive Kit (PE Applied Biosystems) as directed by the manufacturer with the c 10 following modifications: the amount of random primer was cut in half and 5 pl of a gM stock of oligo(dT16) and 5 pl of a 20 unites/pl stock of RNAse inhibitor were added per 100 il reaction. RT reactions were diluted to 50 ng/±l and 5 ng/pl (based on original RNA content) for real time Quantitative PCR (QPCR) analysis. Amounts of specific cis and trans spliced mRNAs were quantitated using Real Time Quantitative PCR. These assays are referred to as Real Time QRT-PCR. These reactions were carried out on the Bio-Rad iCycler iQ Real Time PCR instrument using the SYBR Green kit from PE Applied Biosystems essentially as described previously (Puttaraju et al. 2001 Mol Thr 4:105-14.).

Total HPV-16 RNA levels (cis and trans-spliced) were assessed using a common amplicon in E6 exon 1 (HPV-16 nt 152-204; 53 bp). This assay uses the HPV-16 primers oJMD-15 (ACAGAGCTGCAAACAACTAT) and oJMD-16 (TTGCAGTACACATTCTAA). The amount of RT reaction used for each PCR reaction was 5 ng. Trans-splicing from the HPV-16 nt 226 5' splice site to the PTM lacZ exon was assessed using a 53 bp chimeric amplicon. This assay uses the HPV- 16 senser primer oCCB-348 (GCAAGCAACAGTTACTGCGA; HPV-16 nt 201-220) and the laoZ antisense primer oCCB-322 (ATCCACCCAGTCCCAGA). The amount ofRT reaction used for each PCR reaction was 50 ng. Both assays used the same plasmid (p3671) to generate standard curves for quantitation. Trans-splicing from the HPV-16 nt 880 5' splice site to the PTM lacZ exon was assessed using a 50 bp chimeric amplicon. This assay uses the HPV-16 sense primer oCCB-366 (ATCTACCATGGCTGATCCTG; HPV-16 nt 858-877) and the lacZ antisenser COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:58 FROM- T-966 P088/099 F-541 81 00 0 0 primer oCCB-322. The amount of RT reaction used for each PCR reaction was 00 ng. The plasmid p3672 was used to generate the standard curve for this assay.

Plasmids used as standards for real time QPCR were cloned as follows.

An RT reaction from cotransfections ofp10 5 9 and HPV-PTMI in 293T cells was n 5 used as template for PCR reactions. Primers oCCB-257 (HPV-16 nt 127-147; ACCCAGAAAGTTACCACAGTT) and oCCB-322 gave a 127 bp band which was TOPO-cloned into pCRII-TOPO (Invitrogen) to give p3671 Sequencing showed that C' this DNA corresponds to trans-splicing from HPV-16 nt 226 into the 3' splice site of o the PTM. Primers oJMD-17 (HPV-16 nt 689-708; 10 GACAAGCAGAACCGGACAGA) and oCCB-322 gave a 219 bp band which was TOPO-cloned into pCRII-TOPO to give p3672. Sequencing showed that this DNA corresponds to trans-splicing from HPV-16 at 880 into the 3' splice site of the PTM.

Plasmids stocks (1 ng/pl) were quantitated using PicoGreen (Molecular Probes) prior to use for standard curves.

Quantitation of cis- and trans-splicing for the cotransfections with PTMs and the target lacZCF9m were done exactly as described previously (Puttaraju et al. 2001. Mol. Thor. 4:105-14). The amount ofRT reaction used for each PCR reaction was 5 ng.

13.2 RESULTS HIPV and CF PTMs were contransfected into 293 cells with either the HPV-16 expression vector p1059 to assess trans-splicing efficiency or with lacZCF9m (containing a CF intron) to assess tras-splicing specificity. Real Time QRT-PCR assays were done as described above to assess levels of trans-splicing relative to cis-splicing of each target. The results are shown in Table 1. All RNA levels are expressed as fg of the DNA standard. The standards p36 7 1 and p36 7 2 are close to the same size so these values can be used to represent relative RNA levels for each assays. HPV-PTM1, 2,5, and 6 efficiently trans-spliced to the HPV-16 nt 226 splice site. Up to 70% trans-splicing was seen for the HPV-PTMI. As expected, trans-splicing was abolished by mutations in the branch point and polypyrimidine tracts of the PTM. These PTMs showed less than 1% trans-splicing to COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:59 FROM- T-966 P089/099 F-541 82 00 0 0 1) the nt 880 5' splice site. This data is consistent with the design of these PTMs which 00 have binding domains complementary to the nucleodde 409 and 5263' splice sites.

HPV-PTM-8 and HPV-PTM-9 trans binding domains downstream of the at 880 splice site and show efficient tranzs-splicing to this 5' splice site (37% for TPV-PTMS and 22% for HPV.PTM9) and somewhat less efficient trans-splicing to the ut 226 splice site. PV-PTM9 may interfere stericaly with binding of splicing factors to the nt 880 5' splice site. The specificity ofHPV-PTM1, 2,5 and 6 was also assessedby their ability to tans-splice to a target pre-mRNA with a CF intron. Specificity ranged S274 to 606 fold.

o from 274 to 606 fold.

COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 MSP Co-trmnsfecon 8/2/01 csa Specificity Assay Target p3514 (LaoZCFm) (all cells have target) 6 cm plits; lpciectAmlns, 2C000:2 ug target and 2 ug VIM p3517 for cia splicing standard curve p3519 for ranspllcing standard curre.

SWdt Pri samp Tranafecfon #IPIM i #IAHPV-PTMI 2 #I B HPV-PTM I 3 N2 HPV-PTM2 4 #2B HPV4PTM OA HPV-PTM 6 13B HPV4'TM5 7 #4A HPV-PTMS 8 M4B HPV-PTMS 9 #5A HPV-PTM8 #58 HPV-PTMO 11 MA HPWPTM 12 68 HPV-PTMs 13 #7ACFI4 14 FiB CF14 #8A CF24 169#6 CF24 17 IDA CF27 (OK by PCR) 18 #98 CF27 (OK by PCR) 21 #iA pcDNAS.1 22 #118 pcDNAS.1 23 P3517 10 pg) 24 p351f3l(lpg) .uryu: p3517 uteri: 324123 falCi t200.00 1,80.00 2,840.00 2,090.00 1,820.00 1,750.00 T/2.00 1,720.o 2,570.00 1,800.00 2,30.00 2,660.00 150.00 1,320.00 1,520.00 5soo.oo 1,410.00 1,370.00 1,520.00 1,370.00 0.0 p3519 327322 fe brans I A 3.60 4.10 3.17 1.85 1.82 0.69 2.05 5.06 3.46 2.29 .2.74 22.20 21.50 93.90 7200 3.83 4.53 0.04 0.07 0.17 'Alans 04% 0.3% 0.1% 0.2% 0.1% 0.1 0.1% 0.2% 0.2% 0.1% 0.1% 1.6% 1.6% 5.8% 4.6% 0.3% 023% 0.0% 0.0% NoW: the ldentty f he CF PTMs wa rechecked by PC R of both th plauinkda and the RNJA samples (before ONAs treament) an we OK.

SOOZ q;)j 8 1 6§69ZZOOW 18-02-'08 14:59 FROM- T-966 P091/099 F-541 84 00 0

O

S14. EXAMPLE: DESIGN OF TARGETED PAPILLOMA VIRUS 00 PTMs Initial pre-therapeutic RNA molecules ('PTMs") are developed based on the abundance and splicing patterns of HPV mRNA. The transcription map of 5 HPV-16 in benign infections is shown in Figure 48. Cis and trans splicing assays are NO performed on the initial PTMs and the data obtained from the assays is used to create C' specific PTMs with optimized efficacy in spliceosome-mediated RNA trans-splicing Sreactions.

SThe most effective PTM is one that trans-splices an HPV target transcript with a PTM encoding a toxic product which will kill the infected cell. In targeting the most frequently used HPV splice sites, two viable 5' splice site targets and two viable 3' splice site targets can be used. Less frequently used splice sites can also make good targets if the PTM is designed to block the more frequently used site.

Choice of target splice sites is further restricted if the intention is to treat cancers, since integration of HPV-16 in many cervical cancers leads to expression of only the E6 and E7 regions in these cancers.

The following target splice sites are used in the development of the initial PTMs which leads to the expression of a toxic prodict: i) S' splice site targets: nt 226: This splice site is used in the synthesis of all E6* species.

In most tumors and cell lines, the vast majority of P97 promoter transcripts will be spliced using this 5' splice site.

nt 880: This splice site is used in the synthesis of all E6US (unspliced) and E6* species except E6*11, both splice sites are good targets in both productive infections and cancers; and ii) 3' splice site targets: nt 409: This 3' splice site is used in the splicing of B6*I species which are generally more abundant than E6*II species. This splice site is used in cancers and productive HPV infection.

COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:59 FROM- T-966 P092/099 F-541 00 0 0 S- nt 3358: This target is used for splicing of most mRNAs, but only if 00 the viral DNA is extrachromosomal. This splice site is not a good target for the treatment of most cancers.

In addition, a double trans-splicing PTM is developed to replace the On 5 internal exons nt 409-880 or nt 526-880 in productively infected tissue and in cancers.

SAlternatively, initial PTMs are designed in which trans-splicing produces an mRNA encoding a fusion protein that is part viral and part exogenous c'i peptide encoded by the PTM. The fusion protein will change the function of the viral o protein so that it inhibits an essential viral function. The splice sites listed above are c 10 targeted to produce three viral fusion proteins: The E6 N terminus, using the nt 226 5' splice site as the target; (ii) The E6 C terminus, using the nt 409 (best) or nt 526 3' splice sites as the targets; and (iii) The E2 C terminus, using the nt 3358 3' splice site as target This fusion protein is produced in productive infections and cancers containing extrachromosomal viral DNA. The C terminal domain of E2 is the DNA binding and dimerization domain, and can be used to target a fusion protein to the P97 promoter and block transcription. At high concentrations, the E2 viral protein binds just upstream of the P97 promoter and inhibits transcription by competing with the transcription factors, SP1 and TFIID, for binding. However, these E2 binding sites are weaker than those upstream in the Long Control Region (LCR) and are only saturated at high concentrations of the viral E2 protein. At low concentrations of E2, the protein binds to the E2 binding sites in the upstream LCR and activates transcription. Thus, a "repressor" domain can be added to the fusion protein resulting in a block of transcription through binding to any E2 binding site. This fusion protein is also useful to block viral DNA replication, since an EI/E2 complex binds the origin of replication. It has been demonstrated, however, that a complex of the E2 DNA binding domain and El does not bind to the origin. Since E2 is a dimer, heterodimerization of the E2 fusion protein with full length E2 protein would probably eliminate B2 function in DNA replication.

COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:59 FROM- T-966 P093/099 F-541 86 00 0 0 SPTM(s) based on their ability to target and traits-splice to the HPV T target splice sites depicted in Figure 48 listed above are constructed and screened such 00 that splicing results in the expression of diphtheria toxin sub unit A (DT-A) product, which will kill the infected cells or express a marker gene which can be easily detected. Other peptide or protein toxins may also be encoded. A typical prototype PTM trans-splicing) consists of an antisense target binding domain (25 or more) 0 complementary to HPV sequences, spacer sequence, canonical branchpoint sequence C' (UACUAAC), an extensive polypyrimidine tract (12-15 AG dinucleotide of the O 3' splice site followed by the delivered gene. PTMs are also constructed to carry out C, 10 PTM-mediated trans-splicing with HPV 3' splice sites (Fig. 66B). The trant-splicing domain (TSD) of the PTMs are constructed in modular fashion. Unique restriction sites are incorporated between each of the PTM elements, facilitating the replacement of individual elements. Schematic diagrams of 3' exon replacement and 5' exon replacement models are shown (Figure 66A-B), respectively. It has previously been demonstrated that both efficiency and specificity of trans-splicing can be modulated substantially by altering several sequences in the TSD, including, the length of the binding domain, spacer sequences, strength of the PPT etc.

"Linear" PTMs are designed initially to maximize the trans-splicing efficiency, thereby identifying the PTM sequences that provide highest trans-splicing efficiency. Linear PTMs refer to the binding domain in the PTM as single stranded in configuration To achieve a higher degree of targeting specificity, another form of TSD referred to as a "safety stem" can be constructed. In these PTMs, the splice site of the PTM is protected from reacting with other pre-mRNA targets by binding to itself in a folded structure. Contact with the specific target promotes unwinding of the safety stem exposing and activating the PTM's 3' splice site for spliceosome formation.

To further enhance the trans-splicing specificity, a PTM that requires two trans-splicing events to produce the expected therapeutic effect is also constructed (Fig. 65). This PTM will have an upstream 3' splice site that will transsplice into an HPV 5' splice site, producing a singularly trans-spliced product. This product does not contain the required polyadenylation signals and would be inactive COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 14:59 FROM- T-966 P894/099 F-541 87 00 0 0 0) due to failure in nucleocytoplasmic transport and translation of the mRNA. A second Strans-splicing event with a HPV 3' splice site is necessary to provide the PTM with the signals required for polyadenylation (Fig. 65). Polyadenylation is necessary for PTMs with linear binding domains in which both 3' and 5' binding domains are linear, or with 3' safety 5' linear binding domains, are also designed for inhibition of viral N\ expression. In addition, PTMs are designed as "double safety" PTMs with both 3' and safety splice sites or 3' linear or 5' safety sites.

cl Testing of the PTMs is performed using in vitro splicing assays and O cell culture-based assays. HPV-16-containing cell lines are used for testing the ability C 10 of PTMs to trans-splice. W12 cells (80263 cells) contain extrachromosomal HPV-16 DNA and express the full HPV-16 early region and can be used to test PTMs targeting. SiHa and CaSki cell lines contain integrated HPV-16 and express only the viral B6/E7/5E1 regions. These cell lines are useful because they express viral prenmRNAs characteristic of those expressed in cervical cancers. However, they may not be useful cell lines for testing a PTM targeting the nt 3358 3' splice site. CaSki cells express considerably higher levels of HPV-16 mRNAs than any of the other cell lines tested and therefore may be the best cells for assaying other PTMs.

Cell culture-based cotransfection experiments with a PTM expression vector and an HPV-16 early region expression vector are assayed for expression of the PTM. Several plasmids driving the expression of HPV-16 have been constructed.

For example, two plasmids that can be used in co-transfection experiments include ones that express HPV-16 under the direction of either the SV40 early promoter (p1059) or the K14 promoter (p2571; pK14-1203).

Combined isoform-specific splice-specific) primers with quantitative real time reverse transcription polymerase chain reaction (QRT/PCR) are used to assay for alternative splicing. This assay is very isoform specific, relatively insensitive to RNA degradation, sensitive to one molecule of cDNA, has a wide dynamic range (at least seven orders ofmagnitude), and gives absolute quantitation of each isoform. Primer pairs specific for each PTM/target pre-mRNA combination are used. The sequence specificity of the assay permits the monitoring of the specificity of the trans-splicing reactions. The sensitivity and quantitative nature of the assay as COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 15:00 FROM- T-966 P095/099 F-41 88 00 0 0 Swell as the rapidity with which assays can be developed and performed is useful for 00 the optimization of PTMs targeted against papillomaviral pre-nRNAs.

The specificity ofPTM induced trns-splicing to determine the specificity of targeted trans-splicing to HPV target pre-mRNA) is also evaluated by k) 5 and/or 3' rapid amplification of cDNA ends (RACE) according to stat.dad procedures. This method is relatively fast compared to the conventional cDNA library construction, and gives complete sequences of 5' and/or 3' cDNA ends, so that the number of specific and non-specific splicing events can be determined. Initially, two o cDNA libraries are constructed comprised of RNA isolated from cells co-transfected with a linear PTM HPV mini-gene target and (ii) safety PTM HPV mini-gene target. For example, in order to identify the 5' ends of the trans-spliced RNAs (3' exon replacement), a 5' RACE assay is performed with a PTM antisense primer.

Similarly, to identify the 3' ends of the trans-spliced RNAs exon replacement), a 3' RACE assay is performed using a PTM sense primer. The cDNA is amplified by PCR, digested with restriction enzymes and cloned into a plasnid vector. The oDNA clones are initially screened by colony hybridization using aPTM specific probe.

From each cDNA 'brary, positive clones are selected and sequenced, and the sequence information is used to compare the specificity of linear vs. safety PTM.

This permits identification of non-specific targets that trans-splice at high frequencies. Analysis of these targets provides useful information about the sequences that are responsible for non-specific trans-splicing and helps in the construction of specific PTMs.

The trans-splicing efficiency and specificity data obtained from the analysis of the initial candidate PTMs in trans-splicing assays is used to formulate and develop PTMs with optimal frans-splicing capabilities. The optimal PTMs are analyzed using the trans-splicing assays described above.

A mouse model for papillomavirus infections using an organotypic "raft/xenograft" technique. Papillomaviruses are generally species and cell type specific. Productive infections have been established in nude mice using bovine keratinocytes and bovine papillomaviruses. In this system, keratinocytes are initially plated onto a collagen "raft" containing fibroblasts and allowed to grow to confluence COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 15:00 FROM- T-966 P096/099 F-541 89 00 0 0 in tissue culture. The keratinooytes are then infected or transfected with 00 papillomavirus or viral genomic DNA, respectively, and allowed to grow in culture for a few days. These raft are then grafted onto the backs of nude mice where they develop into productively infected bovine tissue. Human papillomavirus infections can be established using the same techniques combined with human papillomaviruses and keratinocytes. This system is useful for testing the in vivo efficacy of antipapillomavirus PTMs. In addition, grafting of cervical carcinoma tissue or cervical C' cancer cell lines onto nude mice is used. In addition, testing can be done using o several animal models including bovine papillomavirus (BPV-1). Canine oral papillomavirus (COPV), and Cottontail rabbit papillomavirus (CRPV). COPV, in particular, has served as a good model for vaccine development.

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying Figures. Such modifications are intended to fall within the scope of the appended claims. Various references are cited herein, the disclosure of which are incorporated by reference in their entireties.

COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18

Claims (5)

18-02-'08 15:00 FROM- T-966 P097/099 F-541 WiORNLOPrropoMl An flSdeul22150 ppek ditd4,C-2j 14M 00 S o THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS: 00 1. A nucleic acid molecule recognized by nuclear splicing components within a cell comprising: a) one or more target-binding domains, at least one said target-binding domain being between 10 and 600 nucleotides long, wherein said one or more target u binding domains binds the nucleic acid molecule to a target pre-mRNA C expressed within the cell; O b) a 3' splice region comprising a branch point, a pyrimidine tract and a 3' C splice acceptor site and/or a 5' splice site; c) a spacer region that separates the 3' splice region or the 5' splice site from the target-binding domain; and d) nucleotide sequence trans-spliced to the target pre-mRNA to correct a genetic defect in a human Factor VIII gene, wherein a functional clotting factor is produced. 2. The nucleic acid molecule according to Claim 1, wherein the nucleic acid has the 3' splice region and further comprises a 5' donor site. 3. The nucleic acid molecule according to Claim 1 or 2, wherein the nucleic acid further comprises sequence encoding a translatable protein product. 4. The nucleic acid molecule according to Claims 1, 2 or 3, wherein the target pre- mRNA is a Factor VIII encoding pre-mRNA. The nucleic acid molecule according to any preceding Claim, wherein the nucleic acid further comprises nucleotide sequence containing a translational stop codon. 6. The nucleic acid molecule according to Claim 1, wherein the sequence to be trans- spliced replaces exons 23 to 26 of the human Factor VIII gene. 7. The nucleic acid molecule according to any preceding Claim, wherein the nucleic COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 15:0 FROM- T-966 P098/099 F-541 rOf t'rPQroosAalMrdsllCaOe225 o.w2 5 daiAa.c-2200B 00 91 0 acid further comprises a safety sequence, wherein said safety sequence comprises one or more complementary sequences that bind to one or both sides of any one of 00 the 3' splice region or 5' splice site. 8. A cell comprising a recombinant vector recognized by nuclear splicing Scomponents within the cell and which expresses a nucleic acid molecule comprising: a) one or more target-binding domains, at least one said target binding domain Sbeing between 10 and 600 nucleotides long wherein said one or more target binding domains binds the nucleic acid molecule to a target pre-mRNA expressed within the cell; b) a 3' splice region comprising a branchpoint, a pyrimidine tract and a 3' splice acceptor site or a 5' splice site; c) a spacer region that separates the 3' splice region or the 5' splice site from the target-binding domain; and d) nucleotide sequence to be trans-spliced to the target pre-mRNA to correct a genetic defect in a human Factor VIII gene, wherein a functional clotting factor is produced. 9. The cell according to Claim 8, wherein the nucleic acid molecule is as defined in any one of Claims 2 to 7. A method of producing a chimeric RNA molecule in a cell, comprising contacting a target pre-mRNA expressed within the cell with a nucleic acid molecule recognized by nuclear splicing components within the cell and comprising: a) one or more target-binding domains, at least one said target-binding domain being between 10 and 600 nucleotides long wherein said one or more target binding domains binds the nucleic acid molecule to a target pre-mRNA expressed within the cell; b) a 3' splice region comprising a branch point, a pyrimidine tract and a 3' splice acceptor site and/or a 5' splice site; COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 15:00 FROM- T-966 P099/099 F-541 IOtMOtogt\Nnd Antmnaiul\122510 pmax d himWd64S1ii 00 0 S92 c) a spacer region that separates the 3' splice region or the 5' splice site from the 00 target-binding domain; and d) nucleotide sequence trans-spliced to the target pre-mRNA to correct a genetic defect in a human Factor VIII gene, n wherein a functional clotting factor is produced. V\ 11. The method according to Claim 10, wherein the nucleic acid molecule is as defined in any one of Claims 2 to 6. 12. An expression vector which expresses in a cell a nucleic acid molecule claimed in Claim 1, 2 or 3, which is recognized by nuclear splicing components within the cell and comprises: a) one or more target-binding domains, at least one said target-binding domain being between 10 and 600 nucleotides long wherein said one or more target binding domains binds the nucleic acid molecule to a target pre-mRNA expressed within the cell; b) a 3' splice region comprising a branch point, a pyrimidine tract and a Y splice acceptor site and/or a 5' splice site; c) a spacer region that separates the 3' splice region or the 5' splice site from the target-binding domain; and d) nucleotide sequence trans-spliced to the target pre-mRNA to correct a genetic defect in a human Factor VIII gene, wherein a functional clotting factor is produced. 13. The expression vector according to Claim 12, wherein the nucleic acid molecule is as defined in any one of Claims 2 to 7. 14. The expression vector according to Claim 12 or 13, wherein the cell is a eukaryotic cell. A vector for use in gene therapy, which is recognized by nuclear splicing COMS ID No: ARCS-179660 Received by IP Australia: Time 15:01 Date 2008-02-18 18-02-'08 15:01 FROM- T-967 P001/094 F-543 P:OPfl\DWD\Paopo~6 A(&AdwW&IJ2aOc Vprvd da,2/11-2flfoo 00 093 components within a host cell in vivo, or ex vivo, within an autologous host cell for introduction in vivo, wherein the nucleic acid comprises 00 a) one or more target-binding domains, at least one said target-binding domain being between 10 and 600 nucleotides long, and binds the nucleic acid molecule to a target pre-mRNA expressed within the cell; b) a 3' splice region comprising a branch point, a pyrimidine tract and a 3' splice acceptor site and/or a 5' splice site; c, c) a spacer region that separates the 3' splice region or the 5' splice site from the 8target-binding domain; and C, d) nucleotide sequence trans-spliced to the target pre-mRNA to correct a genetic defect in a human Factor VIII gene, wherein a functional clotting factor is produced. 16. A vector according to Claim 15, for use in gene therapy, wherein the cell is a host cell in vivo or is ex vivo an autologous host cell for introduction in vivo. 17. A vector according to Claim 15 in the form of a viral vector. 18. A vector according to Claim 16 which is an adeno-associated virus (AAV), adenovirus or a defective or attenuated retrovirus vector.

19. A composition comprising a pharmaceutically acceptable carrier and a nucleic acid molecule claimed in Claim 1, 2 or 3. A nucleic acid molecule according to Claim 1, 2 or 3 for introducing into target cells of a human subject expressing a human Factor VIii target pre-mRlNA to correct a genetic defect in factor VIII and thereby produce a functional clotting factor.

21. A nucleic acid molecule or vector according to Claim 8 wherein the introduction is indirect, in which host cells are first transformed with the nucleic acid in vitro arid COMS ID No: ARCS-179664 Received by IP Australia: Time 15:09 Date 2008-02-18 18-02-'08 15:01 FROM- T-967 P002/094 F-543 00 then transplanted into the target cells of the subject. 00 22, A nucleic acid molecule or vector according to Claim 8 wherein the introduction is direct, in vivo, ini which the target cells of the subject are directly exposed to the ON nucleic acid.

23. A vector according to claims 13, 14 or 15 for introducing into target cells of a human subject expressing human Factor VIII target prc-mRNA to correct a genetic o defect in factor VIII and therby produce a functional clotting factor.

24. A nucleic acid molecule according to any one of Claims I to 7 or 20 to 22, or a cell according to any one of Claims 8 to 9 or a method according to any one of Claims 10 to I11 or an expression vector according to any one of Claims 12 to 14 or a vector according to any one of Claims 15 to 18 or 23 or a composition according to Claim 19 substantially as hereinbefore described with reference to the ]Figures and/or Examples. COMS ID No: ARCS-i 79664 Received by IP Australia: Time 15:09 Date 2008-02-18