The present invention provides methods and compositions for generating novel nucleic acid molecules through targeted spliceosome mediated simple or double trans- splicing. The compositions of the invention include pre-trans-splicing molecules (PTMs, herein also called "TS molecule" for "Trans-Splicing molecules") designed to interact with a target precursor messenger RNA molecule (target pre-mRNA) and to mediate a simple or double trans-splicing reaction resulting in the generation of a novel chimeric RNA molecule (chimeric RNA). This approach enables to replace whole nucleotide sequences such as exonic sequences in a targeted mRNA and is therefore very interesting to address disorders caused by dominant mutations while preserving levels and tissue specificity. This RNA repair strategy is thus useful to replace mutated nucleic acid sequences into the normal ones and thereby treat many genetic disorders. In particular, the PTMs of the present invention include those genetically engineered to interact with DMD target pre-mRNA so as to result in correction of DMD genetic defects responsible for the Duchenne muscular dystrophy (DMD).

The compositions of the invention further include recombinant vector systems capable of expressing the PTMs of the invention and cells expressing said PTMs. The methods of the invention encompass contacting the PTMs of the invention with a DMD 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 mRNA molecule wherein the genetic defect in the DMD gene has been corrected. The methods and compositions of the present invention can be used in gene therapy for correction of neuromuscular disorders such as the Duchenne muscular dystrophy. The principle of this treatment can also be applied to any genetic disease where the pathogenic mutation involves an alteration of the transcript that can be corrected by simple or double trans-splicing. BACKGROUND OF THE INVENTION

DNA sequences in the chromosome are transcribed into pre-mRNAs which contain coding regions (exons) and generally also contain intervening non-coding regions (introns). Introns are removed from pre-mRNAs in a precise process called cis- splicing. Splicing takes place as a coordinated interaction of several small nuclear ribonucleoprotein particles (snRNPs) and many protein factors that assemble to form an enzymatic complex known as the spliceosome (Staley and Guthrie, 1998).

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 and subsequently in nematodes, flatworms and in plant mitochondria, drosophila, mice an humans (Takayuki Horiuchi and Toshiro Aigaki, 2006).

The mechanism of splice leader 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 2'-5'phosphodiester bond producing a Ύ' 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-splicing reaction, closely resemble their counterparts involved in cis-splicing. 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 immunoglobulin variable region sequence linked to the endogenous constant region in a transgenic mouse (Shimizu et al., 1989). In addition, trans-splicing of c-myb pre-RNA has been demonstrated (Vellard, M. et al. 1992) and more recently, RNA transcripts from cloned SV40 trans- spliced to each other were detected in cultured cells and nuclear extracts (Eul et al., 1995). However, naturally occurring trans- splicing of mammalian pre-mRNAs is thought to be a rare event (Finta, C. et al., 2002). In vitro trans-splicing has been used as a model system to examine the mechanism of splicing by several. Reasonably efficient trans-splicing (30% of cis- spliced analog) was achieved between RNAs capable of base pairing to each other, whereas 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 for example by Chiara & Reed (1995, Nature). These reactions occur at relatively low frequencies and require specialized elements, such as a downstream 5'splice site or exonic splicing enhancers.

The present invention relates to the use of targeted trans-splicing mediated by native mammalian splicing machinery, i. e., spliceosomes, to reprogram or alter the coding sequence of a targeted m-RNA.

A lot of studies have already described PTMs that can mediate "simple" trans- splicing, that is, a technology that enables to replace either the 3' part of a transcript, or, more rarely, the 5' part (Mansfield et al, 2003; Kierlin-Duncan & Sullenger, 2007). For example, U. S. Patent Nos. 6,083, 702,6, 013,487 and 6,280, 978 describe the use of PTMs to mediate a "simple" trans-splicing reaction by contacting a target precursor mRNA to generate novel chimeric RNAs. Importantly, the "simple" trans-splicing technologies enable to correct a number of mutations using minigenes or endogenous transcripts in genetic disease context like hemophilia A (Chao et al., 2003), spinal muscular atrophy (Coady et al., 2008), X-linked immunodeficiency (Tahara et al., 2004) and cystic fibrosis where the widespread mutation CFTRAF508 was replaced efficiently in vivo by the normal sequence via a trans-splicing reaction (Liu et al., 2005). As opposed to "simple" trans-splicing, "double" trans-splicing enables to replace or introduce a sequence, such as exonic sequences, in a targeted mRNA (herein called Exchange: concomitant 3' and 5' trans-splicing reactions). More precisely, double trans-splicing can modify a given or replace a missing exonic sequence within a given gene transcript while at the same time preserving the regulatory intronic sequences which are present 5' and 3' of the exonic sequence targeted by double trans-splicing, thereby allowing for alternative transcripts to occur. Conversely to conventional gene therapy, "double" trans-splicing approaches would be very interesting to address disorders caused by dominant mutations, while preserving levels and tissue specificity. Exon exchange (Exchange) using double trans-splicing, at both sides of a targeted exon, would have the advantage of minimizing exogenous material as well as preserving full regulatory elements potentially present in 5' and/or 3' untranslated domains of the rescued mRNA. As a RNA repair strategy, the Exchange approach will produce the corrected protein where it is naturally expressed. It has the supplementary advantage upon other RNA surgery strategies of correcting precisely the sequence defect without changing anything to the whole messenger sequence (i.e. the open reading frame and untranslated regions). Hence, the regulatory sequences present in 5' and 3' UTRs are preserved, something which never happens in classical gene therapy where cDNAs are amputated of their non coding sequences. These regions are now known to be essential for mRNA stability and translation regulation; in particular, they are targets for miRNAs which play important role in a variety of disease (Zhang & Farwell, 2008).

However no study has ever described so far an efficient PTM enabling to perform a "double" trans-splicing, that is a PTM containing both a 3' splice region and a 5' splice region and a nucleotide sequence to be inserted into a target mRNA. Furthermore, no study has ever demonstrated that such PTM might be able to replace with high efficiency a nucleotide sequence inside a target mRNA.

The herein presented results demonstrate for the first time an efficient PTM enabling high level of Exchange (i.e. concomitant 3' and 5' trans-splicing reactions), and therefore the possibility of rescuing mutated transcripts by specifically replacing a mutated exon by its normal version during a double trans-splicing reaction. By using the PTM presented in the present invention, one can obtain an Exchange efficiency that reaches a level of at least about 50%, for example 53%, of repaired transcripts with DMD minigene as target. Importantly, no non-specific end products were ever detected, suggesting that the chosen annealing sequences of the PTM did not decipher cryptic splicing site nor obstruct splicing events.

The trans-splicing technology uses a trans-splicing molecule that "tricks" the spliceosome into using it as a substrate for splicing. In the Exchange approach, the game is more "tricky" since the spliceosome must realize a double trans-splicing between the pre-messenger transcript and the PTM. After having tested various combinations of antisenses, it was found by the present inventors that the dogmas: i) blocking endogenous splicing signals on the nascent pre-mRNA transcript via base- pairing or ii) at the opposite, bringing the replacing exon closer to endogenous splice site to be joined, did not produce the best results, as shown in the present examples. Indeed, the best antisense for the first trans-splicing (3' replacement) matched with the middle of the first intron while the second one (5' replacement) was better when located close to the 3' end of the exon to be replaced. FIGURE LEGENDS

The figure 1 exposes the trans-splicing strategy for the 3' replacement:

(A) Exons 22 to 24 (boxes E22 to E24) with natural introns (lines with black balls illustrating the splice sites) on the DMD minigene. The cross represents the nonsense mdx mutation in E23. The trans-splicing (TS) molecule AS-E24 comprises a 150nt antisense sequence (AS) complementary to intron 22 as well as a spacer, a strong conserved yeast branch point sequence, a polypyrimidine tract, a 3' acceptor site (the three last elements are represented as blacks balls) and E24. TS constructs were made with three different antisense sequences, AS1 to AS3. Arrows indicate the positions of the forward A and reverse B PCR primers in the minigene and the TS molecule. (B) Expected transcripts generated by cis-splicing (E23 inclusion and skipping) and trans-splicing, and the predicted sizes of the corresponding PCR amplification products detected using the RT-PCR strategy illustrated in (A). (C) RT- PCR analysis using PCR primers A and B of NIH3T3 cells cotransfected with DMD minigene and constructions pSMD2-GFP (Ctrl), pSMD2-U7-SD23-BP22 (U7), pSMD2- E24 (AS-), pSMD2-AS1 -E24 (AS1 ), pSMD2-AS2-E24 (AS2) and pSMD2-AS3-E24 (AS3). RT- AS2: samples containing DMD minigene and pSMD2-AS2-E24 without reverse transcription; H20: PCR negative control. (D) An exact E22-E24 junction was confirmed by sequencing of the 310bp product.

The figure 2 shows the exon replacement approach on DMD reporter transcripts.

(A) The exon exchange molecule (EX) AS-E24-AS' comprises the same elements as the TS molecule (see Fig. 1A) followed by a 5' donor site (black ball) and a second antisens sequence (AS') of 150 nt complementary to intron 23. EX constructs were made with five different AS' antisense sequences, AS4 to AS8. Arrows indicate the positions of the forward A and reverse C PCR primers in the minigene. (B) Expected transcripts generated by cis-splicing (E23 inclusion and skipping) and exon exchange, and predicted sizes of the corresponding PCR amplification products detected using the RT-PCR strategy illustrated in (A). (C) RT-PCR analysis using primers A and C of NIH3T3 cells cotransfected with DMD minigene and constructions pSMD2-GFP (Ctrl), pSMD2-U7-SD23-BP22 (U7), the TS constructions pSMD2-AS1 -E24 (AS1 ), pSMD2- AS2-E24 (AS2) and EX molecules pSMD2-AS-E24-AS' containing AS1 or AS2 and AS4 to AS8. AS2-2XAS4, EX plasmid pSMD2-AS2-E24-2XAS4 containing two AS4 copies; H20: PCR negative control. (D) Accurate E22-E24 and E24-E24 junctions were confirmed by sequencing of the 408bp product.

The figure 3 shows the effect of intronic splice enhancer sequences on exon replacement efficiency.

(A) Exon exchange molecules AS-E24-AS' with intronic splice enhancers ISE or DISE sequences. (B) RT-PCR analysis using primers A and C of NIH3T3 cells cotransfected with DMD minigene and constructs pSMD2-GFP (Ctrl), pSMD2-U7-SD23-BP22 (U7) and the following EX plasmids with AS4 or AS8: pSMD2-AS2-E24-AS' (-), pSMD2- AS2-ISE-E24-AS' (ISE), pSMD2-AS2-E24-DISE-AS' (DISE) and pSMD2-AS2-E24- 2XAS' (2XAS'). H20: PCR negative control. (C) Efficiency of DMD exon exchange induced by AS4 containing EX molecules analyzed by absolute quantitative real-time RT-PCR.

The figure 4 shows the trans-splicing strategy for dystrophin transcript repair with the simple transsplicing molecule of the invention SEQ ID NO 71 (example 2)

The endogenous pre-messenger dystrophin transcript is illustrated on the top with boxes representing exons, and black lines representing introns. The trans-splicing mRNA molecule (second line) comprise a 150nt antisense sequence complementary to intron 58 of the DMD gene as well as a spacer, a strong conserved yeast branch point sequence (BP), a polypyrimidine tract (PPT), a 3' splice acceptor (SA) and the normal human dystrophin cDNA from exon 59 to the exon 79 STOP codon.

The figure 5 shows the detection of repaired dystrophin transcripts with the simple transsplicing molecule of the invention SEQ ID NO 71 (example 2)

(A) Mutated dystrophin mRNA is represented on the top, as well as the trans-splicing mRNA molecule SEQ ID NO: (second line), and expected dystrophin transcripts generated by cis-splicing and trans-splicing (third line). Arrows indicate the positions of the forward Fo/Fi and reverse Ro/Ri PCR primers designed to detect only the repaired dystrophin cDNA by generating a 2443bp PCR product. (B) RT-PCR analysis using PCR primers Fo/Fi and Ro/Ri of total RNAs extracted from patient myotubes (DMD) transduced by lentivirus expressing the simple trans-splicing mRNA molecules (TsM). Lane DMD non transduced myotubes.

The figure 6 shows the Dystrophin rescue in DMD cells using the simple transsplicing molecule of the invention SEQ ID NO 71 (example 2)

Western blot of total protein extracted from DMD patient myotubes transduced by lentivirus expressing the TSM molecules, stained with the NCL-DYS1 monoclonal antibody. The full-length 427 kD dystrophin is indicated as detected in normal CHQ myotubes sample (WT). Lane DMD non transduced myotubes. Each lane was loaded with 50 μg of total protein except Ctrl, 5μg. Red panel: visualization of total proteins present on the same membrane by Ponceau red staining.

SUMMARY OF THE INVENTION

In a first aspect, the present invention is drawn to a nucleic acid molecule comprising: a) two target binding domains AS and AS' that target the binding of the nucleic acid molecule to a target pre-mRNA, wherein the two target binding domains AS and

AS' are located respectively at the 5'-end and at the 3'-end of the nucleic acid molecule, b) a 3' splice region comprising a branch point, a polypyrimidine tract and a 3' splice acceptor site, c) a 5' splice region comprising a 5' splice donor site, d) a spacer sequence that separates the 3' splice region from the 5'-end target binding domain AS, e) a spacer sequence that separates the 5' splice region from the 3'-end target binding domain AS', and f) a nucleotide sequence to be irans-spliced to the target pre-mRNA, wherein said nucleotide sequence encodes at least a part of a normal polypeptide, and is located between the 3' splice region and the 5' splice region of said nucleic acid.

This nucleic acid molecule is hereafter referred to as "double trans-splicing molecule", or "double PTM" of the invention.

Preferably, said nucleic acid molecule comprises: a) two target binding domains AS and AS' that target binding of the nucleic acid molecule to the pre-mRNA of the dystrophin gene (DMD), wherein the two target binding domains AS and AS' are located respectively at the 5'-end and at the 3'-end of the nucleic acid molecule, b) a 3' splice region comprising a branch point, a polypyrimidine tract and a 3' splice acceptor site, c) a 5' splice region comprising a 5' splice donor site, d) a spacer sequence that separates the 3' splice region from the 5'-end target binding domain AS, e) a spacer sequence that separates the 5' splice region from the 3'-end target binding domain AS', and f) a nucleotide sequence to be irans-spliced to the target pre-mRNA of the dystrophin gene (DMD), wherein said nucleotide sequence encodes at least a part of the normal dystrophin polypeptide, and is located between the 3' splice region and the 5' splice region of said nucleic acid.

In a second aspect, the present invention is drawn to a nucleic acid molecule comprising: a) one target binding domain (AS) that target binding of the nucleic acid molecule to the pre-mRNA of the dystrophin gene (DMD), b) a 3' splice region comprising a branch point, a polypyrimidine tract and a 3' splice acceptor site, c) a spacer sequence that separates the 3' splice region from the target binding domain AS, d) a nucleotide sequence to be irans-spliced to the target pre-mRNA wherein said nucleotide sequence encodes at least a part of the DMD polypeptide.

This nucleic acid molecule is hereafter referred to as "simple trans-splicing molecule", or "simple PTM" of the invention.

The present invention is also drawn to a recombinant vector comprising the PTMs of the invention and to a cell comprising the PTMs of the invention, or the recombinant vector of comprising the PTMs of the invention.

The compositions and methods can 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.

Specifically, the compositions and methods can be used to replace in vitro a mutated endogenous exon 23 or exon 70 of the DMD gene within a cell, comprising contacting the cellular pre-mRNA of the DMD gene with the PTMs of the present invention, under conditions in which the nucleotide sequence to be irans-spliced is irans-spliced to the target pre-mRNA of the DMD gene to form a chimeric mRNA within the cell.

To go further, the present invention also discloses a method for in vivo correcting a DMD genetic defect in a subject, comprising administering to said subject the PTMs of the invention, or the vector comprising the PTMs of the invention, or the cell comprising the PTMs of the invention.

More specifically, the present invention discloses a method for correcting at least one genetic mutation present in exon 23 or 70 of the DMD gene in a subject in need thereof, comprising administering to said subject the PTMs of the invention, wherein the nucleotide sequence to be frans-spliced is at least the exon 23 or at least the exon 70 of the DMD gene. The present invention also provides pharmaceutical compositions comprising an effective amount of the PTMs of the invention and a pharmaceutically acceptable carrier.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In a first aspect, the present invention relies on the designing and the optimization of a double PTM dedicated to concomitant 3' and 5' trans-splicing reactions in order to replace specific nucleotide sequence, and for example a mutated exon, or to replace missing exons in the case of deletion mutations. Such a double PTM therefore necessarily contains a 3' splice region and a 5' splice region. Also, the double PTM must contain at least two distinct target binding domains that enable the PTM to recognize and get very close to the target mRNA. In the context of the invention, these two target binding domains are called "AS" (for Anti Sens).

In this first aspect, the present invention is thus drawn to a nucleic acid molecule comprising: a) two target binding domains AS and AS' that target the binding of the nucleic acid molecule to a target pre-mRNA, wherein the two target binding domains AS and AS' are located respectively at the 5'-end and at the 3'-end of the nucleic acid molecule, b) a 3' splice region comprising a branch point, a polypyrimidine tract and a 3' splice acceptor site, c) a 5' splice region comprising a 5' splice donor site, d) a spacer sequence that separates the 3' splice region from the 5'-end target binding domain AS, e) a spacer sequence that separates the 5' splice region from the 3'-end target binding domain AS', and f) a nucleotide sequence to be irans-spliced to the target pre-mRNA, wherein said nucleotide sequence encodes at least a part of a normal polypeptide, and is located between the 3' splice region and the 5' splice region of said nucleic acid. This nucleic acid molecule is hereafter designated by the "double trans-splicing molecule of the invention" or "the double PTM of the invention".

The target binding domain of a PTM endows the PTM with a binding affinity for the target pre-mRNA. As used herein, a target binding domain is defined as any molecule, i. e. , nucleotide, protein, chemical compound, etc., that confers specificity of binding and anchors the pre-mRNA closely in space to the PTM 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 of the PTM are preferably nucleotide sequences which are complementary to and in anti-sense orientation to the targeted region of the selected target pre-mRNA. The target binding domains may comprise up to several thousand nucleotides. In preferred embodiments of the invention the target binding domains may comprise between about 100 and 200 nucleotides, and preferably about 150 nucleotides.

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 target pre-mRNA, forming 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 al., 1989, Molecular Cloning, A Laboratory Manual, 2d Ed. , Cold Spring Harbor 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. Binding domains may encompass any or all sequences located within the target intron and flanking exons and may consist of contiguous sequence or contain sequence gaps ranging in size from a few to several hundred nucleotides in length. In such cases, the binding domain may be considered to be comprised of multiple, smaller binding domains that are positioned within the PTM in either orientation (sense or antisense) relative to the target sequence or to each other. Any or all sequence elements within the binding domain may contain significant complementarity to the target region.

After having tested various combinations of antisens nucleotide sequences, the present inventors have found that, contrary to what was commonly taught in the art, it is no use to block the endogenous splicing signals on the nascent pre-mRNA transcript via base-pairing. Therefore, in a preferred embodiment, the target binding domains of the double PTM of the present invention are not blocking the endogenous splicing signals on the nascent pre-RNA transcript.

In a first embodiment, the target pre-RNA is a mutated exon of a gene, and the nucleotide sequence to be irans-spliced to the target pre-mRNA, is the corresponding normal exon, or exons, of said gene.

The gene targeted by double-trans-splicing must be a gene that is actively transcribed in the cell targeted by the procedure. It must be composed of several exons that are transcribed into a pre-mRNA molecule. In this case, it has been shown here for the first time that bringing the replacing exon closer to endogenous splice site to be joined does not produce the best results. Therefore, in the context of the invention, the target binding domains AS and AS' are preferably not complementary to sequences that are close to the endogenous splice sites of the mutated exon to be replaced, that is, below 200 nucleotides from the endogenous splice sites.

The double PTM molecule also contains a 3' splice region that includes a branch point sequence, a polypyrimidine tract (such as SEQ ID NO 28) and a 3' splice acceptor site. The double PTM molecule also contains a 5' splice region.

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 regions may be used in the practice of the invention. Briefly, the mammalian consensus sequences for the 5' donor splice site and the 3' acceptor splice site are respectively: GTAAGT and TCCCTCCAG. For example, the 5' donor splice site of the double PTM of the invention can be GTAAGA (SEQ ID NO: 30) and the 3' acceptor splice site of the double PTM of the invention can be GGAAAACAG (SEQ ID NO: 29 ).

The branch point consensus sequence in mammals is YNYURAC (Y=pyrimidine; N=any nucleotide; R=purine). For example, the branch point can be TACTAAC (SEQ ID NO :25 ) corresponding to the well conserved yeast branch point (Mansfield et al 2000). The A is the site of branch formation. A polypyrimidine tract is located between the branch point and the 3' splice site acceptor and is important for different branch point utilization and 3' splice site recognition. Recently, pre-mRNA introns beginning with the dinucleotide AU and ending with the dinucleotide AC have been identified and referred to as U12 introns. U12 intron sequences as well as any sequences that function as splice acceptor/donor sequences may also be used to generate the double PTMs of the invention.

A spacer region to separate the RNA splice site from the target binding domain is also included in the double PTM. The double PTM of the invention contains at least two different spacers: a spacer sequence that separates the 3' splice region from the 5'-end target binding domain AS, and a spacer sequence that separates the 5' splice region from the 3'-end target binding domain AS'. They are preferably non coding sequences and comprise between 10 and 100 nucleotides, preferably between 20 and 70 nucleotides, more preferably between 30 and 50 nucleotides. The spacer regions may be designed to include features such as stop codons which would block any translation of a spliced PTM. In an embodiment of the invention, splicing enhancers such as, for example, sequences referred to as exonic splicing enhancers may also be included in the double PTM design. Transacting splicing factors, namely the serine/arginine-rich (SR) proteins, have been shown to interact with such exonic splicing enhancers and modulate splicing (Tacke et al., 1999). Also, the G-rich intronic splice enhancer from the human GH-1 gene (SEQ ID NO: 26) and/or the DISE sequence from the rat FGFR2 gene (SEQ ID NO: 27) can be used as splicing enhancers.

In a more preferred embodiment, the spacer sequence that separates the 3' splice region from the 5'-end target binding domain AS contains a ISE, for example the G-rich intronic splice enhancer from the human GH-1 gene (SEQ ID NO: 26), and the spacer sequence that separates the 5' splice region from the 3'-end target binding domain AS contains a DISE sequence, for example the DISE sequence from the rat FGFR2 gene (SEQ ID NO: 27), preferably in close vicinity of the nucleotide sequence to be trans-spliced, that is the DISE sequence should be located not farer than 60 nucleotides from the nucleotide sequence to be trans-spliced.

Additional features can be added to the double PTM molecule either after, or before, the nucleotide sequence encoding a translatable protein, such as polyadenylation signals to modify RNA expression/stability, 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. In addition, stop codons may be included in the PTM structure to prevent translation of unspliced PTMs. Further elements such as a 3' hairpin structure, circularized RNA, nucleotide base modification, or synthetic analogs can be incorporated into double PTMs to promote or facilitate nuclear localization and spliceosomal incorporation, and intra-cellular stability.

As an example, the present invention relates to the design and the optimization of Exchange constructs (PTMs) designed for rescuing mutated mRNAs from very large genes such as the dystrophin gene DMD (for example the human DMD gene identified as NC_000023.10 or the mouse DMD gene identified as NC_000086.6j. Mutations in the dystrophin gene DMD cause the Duchenne muscular dystrophy (DMD), the most common severe childhood muscular pathology. Recently, exon skipping strategies have proven to be efficacious in restoring functional dystrophin expression in models of muscular dystrophy including the mdx mouse, the GRMD dog and muscle stem cells from DMD patients and in four DMD patients by local intramuscular injection (Goyenvalle et al., 2004; Denti et al., 2006; Yokota et al., 2009). Indeed, the modular structure of the dystrophin, with its central rod-domain made of 24 spectrin-like repeats, tolerates large truncations. However, exon skipping strategies only concern patients for whom forced splicing would generate a shorter but still functional protein. Many pathological situations escape this prerequisite. In this context, Exchange strategies could be of great interest for replacing precisely a mutated exon of DMD by a normal corresponding exon, and for example the mutated exon 23 which carries a stop mutation in the mdx mouse model of DMD (cf. SEQ ID NO 9), or a genetic anomaly present between exons 59 and 79, which represent 8% of Duchenne patients. Furthermore, Exchange strategies could be useful for replacing missing exons, thereby producing a full length gene product instead of a truncated gene product as results from exon skipping approaches.

In such a view, the present invention shows here for the first time a PTM enabling an efficient exon exchange (for example one exon of the DMD gene, and more particularly the exon 23 of the DMD gene) importantly demonstrating that the Exchange of a specific exon is possible and efficient with the "double" trans-splicing technology. In a preferred embodiment, the present invention is thus drawn to a nucleic acid molecule comprising: a) two target binding domains AS and AS' that target binding of the nucleic acid molecule to the pre-mRNA of the dystrophin gene (DMD), wherein the two target binding domains AS and AS' are located respectively at the 5'-end and at the 3'-end of the nucleic acid molecule, b) a 3' splice region comprising a branch point, a polypyrimidine tract and a 3' splice acceptor site, c) a 5' splice region comprising a 5' splice donor site, d) a spacer sequence that separates the 3' splice region from the 5'-end target binding domain AS, e) a spacer sequence that separates the 5' splice region from the 3'-end target binding domain AS', and f) a nucleotide sequence to be irans-spliced to the target pre-mRNA of the dystrophin gene (DMD), wherein said nucleotide sequence encodes at least a part of the normal dystrophin polypeptide, and is located between the 3' splice region and the 5' splice region of said nucleic acid.

The general design, construction and genetic engineering of PTMs and demonstration of their ability to mediate successful trans-splicing reactions within the cell are described in detail in U. S. Patent Nos. 6, 083, 702,6, 013,487 and 6, 280,978 as well as patent Serial Nos. 09/941 , 492,09/756, 095,09/756, 096 and 09/756,097 the disclosures of which are incorporated by reference in their entirety herein.

In a particular embodiment, in the double PTM of the present invention, the nucleotide sequence to be irans-spliced comprises at least one exon of the normal DMD gene, preferably the sequence of exon 23 of the normal DMD gene, that is SEQ ID NO 8 (from the mouse gene) or SEQ ID NO 60 (from the human gene), or the exon 70 of the normal DMD gene (SEQ ID NO 72 for the human gene, SEQ ID NO 73 for the mouse gene). In a preferred embodiment of the invention, the 5'-end target binding domain AS targets the binding of the nucleic acid to the intron 22 of the pre-mRNA of the DMD gene (SEQ ID NO 1 1 for the mouse gene, or SEQ ID NO 61 for the human gene) and the 3'-end target binding domain AS' targets the binding of the nucleic acid to the intron 23 of the pre-RNA of the DMD gene (SEQ ID NO 12 for the mouse gene, SEQ ID NO 62 for the human gene).

In an embodiment of the invention, the target binding domains AS and AS' comprises between about 100 and about 200 nucleotides, preferably about 150 nucleotides. In a preferred embodiment, the 5'-end target binding domain AS comprises at least 20 successive nucleotides of one of the nucleotide sequence chosen among: SEQ ID NO 13 (hereafter called "AS1 ") and SEQ ID NO 14 (hereafter called "AS2"). Preferably, the 5'-end target binding domain AS comprises at least 20 successive nucleotides of SEQ ID NO 15. On the other hand, the 3'-end target binding domain AS' targets preferably the binding of the nucleic acid to a nucleotide sequence located in the 5'-half of the nucleotide sequence of intron 23, and, more preferably, to a nucleotide sequence located in SEQ ID NO 22 (for the mouse gene) and comprises at least 20 successive nucleotides of one of the nucleotide sequences chosen among: SEQ ID NO 16 (hereafter called "AS4"), SEQ ID NO 19 (herafter called "AS 7"), SEQ ID NO 20 (hereafter called "AS8") and SEQ ID NO 21 (hereafter called "2XAS4"). In a preferred embodiment, the 3'-end target binding domain AS' comprises at least 20 successive nucleotides of SEQ ID NO 21 .

In another embodiment, the double PTM of the invention comprises a conserved yeast branch point sequence , for example the yeast branch point of SEQ ID NO 25.

In another embodiment, the spacer separating the 5' splice donor site and the 3'-end target binding domain AS' comprises between 10 and 100 nucleotides, preferably between 20 and 70 nucleotides, more preferably between 30 and 50 nucleotides. In a preferred embodiment, this spacer comprises a downstream intronic splice enhancer (DISE), which is preferably the DISE sequence from the rat FGFR2 gene, i.e. the SEQ ID NO 27. The spacer sequence that separates the 3' splice region from the 5'-end target binding domain AS comprises between 10 and 100 nucleotides, preferably between 20 and 70 nucleotides, more preferably between 30 and 50 nucleotides.

Such spacers are preferably non coding sequences but may be designed to include features such as stop codons which would block any translation of a spliced PTM. Examples of useful spacers are given in the experimental part of this application. They are for example SEQ ID NO 23 (3' end), and SEQ ID NO 24 (5' end).

In a specific embodiment, the present invention is drawn to a recombinant vector comprising the nucleic acid previously described. More particularly, the double PTM of interest may be recombinantly engineered into a variety of host vector systems that also provide for replication of the DNA in large scale and contain the necessary elements for directing the transcription of the double PTM. The use of such a construct to transfect target cells in the patient will result in the transcription of sufficient amounts of double PTMs that will form complementary base pairs with the endogenously expressed pre-mRNA targets, such as for example, DMD pre-mRNA target, and thereby facilitate a trans-splicing reaction between the complexed nucleic acid molecules. Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired RNA, i. e., PTM. Such vectors can be constructed by recombinant DNA technology methods standard in the art. Vectors comprising the double PTM of interest can be plasmid, viral, or others known in the art, used for replication and expression in mammalian cells. Expression of the double PTM can be regulated by any promoter/enhancer sequences known in the art to act in mammalian, preferably human cells. Such promoters/enhancers can be inducible or constitutive. Such promoters include but are not limited to: the SV40 early promoter region, the promoter contained in the 3' long terminal repeat of Rous sarcoma virus, the herpes thymidine kinase promoter, the regulatory sequences of the metallothionein gene, the viral CMV promoter, the human chorionic gonadotropin-P promoter , etc. Any type of plasmid, cosmid, YAC or viral vector can be used to prepare the recombinant DNA construct which can be introduced directly into the tissue site. Alternatively, viral vectors can be used which selectively infect the desired target 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 of retroviruses, adenoviruses or adeno-associated viruses. In a preferred embodiment, the recombinant vector of the invention is an eukaryotic expression vector.

In another specific embodiment, the present invention comprises delivering the double PTM of the invention to a target cell. Various delivery systems are known and can be used to transfer the compositions of the invention into cells, e. g. encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the composition, receptor-mediated endocytosis, construction of a nucleic acid as part of a retroviral, adenoviral, adeno-associated viral or other vector, injection of DNA, electroporation, calcium phosphate mediated transfection, etc. In this case, the 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 double trans-splicing reaction resulting in the formation of a chimeric RNA comprising a portion of the PTM molecule spliced to a portion of the pre-mRNA. The present invention also concerns a cell comprising the double PTM of the invention, or the recombinant vector of comprising the double PTM of the invention.

In a preferred embodiment, the cell comprising the double PTM or the recombinant vector comprising the double PTM is an eukaryotic cell.

The compositions and methods can 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.

Specifically, the compositions and methods can be used to replace in vitro a mutated endogenous exon of the DMD gene within a cell comprising contacting the cellular pre-mRNA of the DMD gene with the double PTM of the present invention, under conditions in which the nucleotide sequence to be irans-spliced is irans-spliced to the target pre-mRNA of the DMD gene to form a chimeric mRNA within the cell. Said mutated exon is preferably exon 23 of the DMD gene.

To go further, the present invention also discloses a method for in vivo correcting a DMD genetic defect in a subject, comprising administering to said subject the double PTM of the invention, or the vector comprising the double PTM of the invention, or the cell comprising the double PTM of the invention. More specifically, the present invention discloses a method for correcting at least one genetic mutation present in at least one endogenous mutated exon of the DMD gene in a subject in need thereof, comprising administering to said subject the double PTM of the invention. Preferably, said endogenous mutated exon is exon 23 of the DMD gene, or exon 70 of the DMD gene, and the nucleotide sequence to be frans-spliced comprises at least the exon 23 of the DMD gene (SEQ ID NO 8 for the mouse gene, SEQ ID NO 60 for the human gene), or at least the exon 70 of the DMD gene (SEQ ID NO 72 for the mouse gene, SEQ ID NO 73 for the human gene). In other words, the present invention discloses a double PTM, a vector comprising it, or a cell comprising it, for their use for correcting a DMD genetic defect in a subject in need thereof, or, more precisely, for their use for correcting at least one genetic mutation present in at least one of the DMD gene in a subject in need thereof, wherein, preferably said endogenous mutated exon is exon 23 of the DMD gene or exon 70 of the DMD gene, and the nucleotide sequence to be irans-spliced comprises at least the exon 23 of the DMD gene (SEQ ID NO 8 for the mouse gene, SEQ ID NO 60 for the human gene), or at least the exon 70 of the DMD gene (SEQ ID NO 72 for the mouse gene, SEQ ID NO 73 for the human gene).

The present invention also provides pharmaceutical compositions comprising an effective amount of the double PTM of the invention, 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 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 administered. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical sciences" by E. W. Martin.

Eventually, the present invention is drawn to a method for treating the Duchenne muscular dystrophy in a subject in need thereof, comprising administering to said subject the pharmaceutical composition comprising the double PTM of the invention. Preferably, the nucleotide sequence to be irans-spliced comprises at least an exon of the DMD gene, for example the exon 23 of the DMD gene (SEQ ID NO 8 or SEQ ID NO 60), or the exon 70 of the DMD gene (SEQ ID NO 72 or SEQ ID NO 73). In other words, the present invention covers the double PTM of the invention for its use for treating the Duchenne muscular dystrophy in a subject in need thereof. Preferably, the nucleotide sequence to be irans-spliced is at least an exon of the DMD gene, for example the exon 23 of the DMD gene (SEQ ID NO 8 or SEQ ID NO 60) or the exon 70 of the DMD gene (SEQ I D NO 72 or SEQ I D NO 73).

In a specific embodiment, it may be desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment, i. e. in the muscles. This may be achieved by, for example, and not by way of limitation, local infusion during surgery, loco-regional infusion under high pressure in a limb where the arterial and venous blood flux is intermittently interrupted by a tourniquet, topical application, e. g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. Other controled release drug delivery systems exist, such as nanoparticles, matrices such as controlled-release polymers, hydrogels. The double PTM will be administered in amounts which are effective to produce the desired effect in the targeted cell. Effective dosages of the double 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 severity of the DMD being treated, and can be determined by standard clinical techniques.

The double PTM can also be delivered to cells or stem cells ex vivo which can, in a second step after ex vivo correction, be transferred as a cell transplant to an individual with the goal of correcting an organ or an individual affected by a genetic disease through cell therapy.

In a second aspect, the present invention is drawn to a nucleic acid molecule dedicated to simple trans-splicing as it is described in the following examples.

In this particular embodiment, the nucleic acid molecule of the invention comprises: a) one target binding domain (AS) that target binding of the nucleic acid molecule to the pre-mRNA of the dystrophin gene (DMD), b) a 3' splice region comprising a branch point, a polypyrimidine tract and a 3' splice acceptor site, c) a spacer sequence that separates the 3' splice region from the target binding domain AS, d) a nucleotide sequence to be irans-spliced to the target pre-mRNA wherein said nucleotide sequence encodes at least a part of the DMD polypeptide.

This nucleic acid molecule is hereafter referred to as "simple trans-splicing molecule", or "simple PTM" of the invention.

The different parts of this simple trans-splicing molecule (3' splice acceptor site, branch point, polypyrimidine tract, spacer sequence) are the same as described above for the PTM mediating double trans-splicing, that is, for example, SEQ ID NO 29 for the 3' splice acceptor site, SEQ ID NO 25 for the branch point, SEQ ID NO 28 for the polypyrimidine tract, SEQ ID NO 23 for the 3' end spacer.

As a matter of fact, the present invention shows also here for the first time a simple PTM enabling efficient exon(s) replacement (for example replacing anormal exon 70 of the human DMD gene, by the normal one), importantly demonstrating that the replacement of specific exon(s) is possible and efficient also with a "simple" trans- splicing technology (cf. example 2).

In this simple PTM, the nucleotide sequence to be trans-spliced to the pre- mRNA of the dystrophin gene (DMD) comprises preferably one or several exon(s) of the normal DMD gene, more preferably the exon 23 or any one of the exons 59 to 79 of the DMD gene. Even more preferably, the sequence to be trans-spliced is the exon 70 of the DMD gene, or the cDNA from all the exons 59 to 79 of the DMD gene, as shown in example 2 below. More precisely, in the simple PTM of the present invention, the nucleotide sequence to be irans-spliced is either the sequence of exon 23 of the normal DMD gene (that is SEQ ID NO 8 for the mouse gene or SEQ ID NO 60 for the human gene), or the sequence of exon 70 of the DMD gene (that is, SEQ ID NO 72 for the human gene, and SEQ ID NO 73 for the mouse gene), or the cDNA corresponding to exons 59 to 79 of the DMD gene (that is SEQ ID NO 69 for the mouse gene or SEQ ID NO 70 for the human gene). In a preferred embodiment of the invention, the 5'end target binding domain AS of the simple PTM of the invention targets the binding of the nucleic acid to intron 22 of the pre-mRNA of the DMD gene (SEQ ID NO 1 1 for the mouse gene, or SEQ ID NO 61 for the human gene), or to intron 58 of the pre-mRNA of the DMD gene (SEQ ID NO: 66 for the mouse gene, SEQ ID NO 67 for the human gene).

In a more preferred embodiment, the 5' end target binding domain AS of the simple PTM of the invention has the sequence SEQ ID NO 68 (for targeting the PTM to intron 58 of the DMD gene) or SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, or SEQ ID NO 58 (for targeting the PTM to intron 22 of the DMD gene). In an even more preferred embodiment, the simple trans-splicing molecule of the invention has the SEQ ID NO 71.

In a specific embodiment, the present invention is drawn to a recombinant vector comprising the simple PTM previously described. More particularly, the simple PTM of interest may be recombinantly engineered into a variety of host vector systems that also provide for replication of the DNA in large scale and contain the necessary elements for directing the transcription of the simple PTM. The use of such a construct to transfect target cells in the patient will result in the transcription of sufficient amounts of simple PTMs that will form complementary base pairs with the endogenously expressed pre-mRNA targets, such as for example, DMD pre-mRNA target, and thereby facilitate a trans-splicing reaction between the complexed nucleic acid molecules. Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired RNA, i. e., PTM. Such vectors can be constructed by recombinant DNA technology methods standard in the art. Vectors comprising the simple PTM of interest can be plasmid, viral, or others known in the art, used for replication and expression in mammalian cells. Expression of the simple PTM can be regulated by any promoter/enhancer sequences known in the art to act in mammalian, preferably human cells. Such promoters/enhancers can be inducible or constitutive. Such promoters include but are not limited to: the SV40 early promoter region, the promoter contained in the 3' long terminal repeat of Rous sarcoma virus, the herpes thymidine kinase promoter, the regulatory sequences of the metallothionein gene, the viral CMV promoter, the human chorionic gonadotropin-P promoter , etc. Any type of plasmid, cosmid, YAC or viral vector can be used to prepare the recombinant DNA construct which can be introduced directly into the tissue site. Alternatively, viral vectors can be used which selectively infect the desired target 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 of retroviruses, adenoviruses or adeno-associated viruses. In a preferred embodiment, the recombinant vector of the invention is an eukaryotic expression vector.

In another specific embodiment, the present invention comprises delivering the simple PTM of the invention to a target cell. Various delivery systems are known and can be used to transfer the compositions of the invention into cells, e. g. encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the composition, receptor-mediated endocytosis, construction of a nucleic acid as part of a retroviral, adenoviral, adeno-associated viral or other vector, injection of DNA, electroporation, calcium phosphate mediated transfection, etc. In this case, the simple 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 simple PTM binds to a pre-mRNA and mediates a simple trans-splicing reaction resulting in the formation of a chimeric RNA comprising a portion of the simple PTM molecule spliced to a portion of the pre-mRNA. The present invention also concerns a cell comprising the simple PTM of the invention, or the recombinant vector of comprising the simple PTM of the invention.

In a preferred embodiment, the cell comprising the simple PTM or the recombinant vector comprising the simple PTM is an eukaryotic cell.

The simple PTM of the invention appears to be a very interesting tool to target DMD patients independently of their DMD mutation. By enabling to restore mutated exons of the DMD gene, this molecule appears to be also an efficient tool to treat DMD patients.

Therefore, the present invention is also drawn to a method for treating a patient suffering from the Duchenne muscular dystrophy comprising administering to said patient a pharmaceutical composition comprising the simple trans-splicing molecule of the invention or the recombinant vector comprising it, or the cell comprising them. In other words, the present invention is drawn to the simple trans-splicing molecule of the invention, or the recombinant vector comprising it, or the cell comprising them for their use in a pharmaceutical composition for treating the Duchenne muscular dystrophy in a subject in need thereof.

More precisely, the present invention also concerns a method for correcting a DMD genetic defect in a subject, comprising administering to said subject the simple trans-splicing molecule of the invention, or the recombinant vector comprising it, or the cell comprising them. In other words, the invention is drawn to the simple trans-splicing molecule of the invention, or the recombinant vector comprising it, or the cell comprising them, for their use for correcting a DMD genetic defect in a subject in need thereof. Preferably, the nucleotide sequence to be frans-spliced comprises at least one exon of the DMD gene, and comprises more preferably at least exon 23 of the normal DMD gene (that is SEQ ID NO 8 for the mouse gene or SEQ ID NO 60 for the human gene), or exon 70 of the DMD gene (that is, SEQ ID NO 72 for the human gene, or SEQ ID NO 73 for the murine gene) or any exon chosen among exons 59 to 79 of the DMD gene. In a preferred embodiment, it comprises the cDNA from exon 59 to 79 of DMD gene (that is, SEQ ID NO 70 for the human gene, and SEQ ID NO 69 for the mouse gene).

The present invention also concerns an in vitro method of replacing at least one mutated endogenous exon of the DMD gene within a cell, comprising contacting the cellular pre-mRNA of the DMD gene with the simple trans-splicing molecule of the invention, under conditions in which the nucleotide sequence to be irans-spliced is irans-spliced to the target pre-mRNA of the DMD gene to form a chimeric mRNA within the cell. Preferably, said mutated exon is exon 23 of the DMD gene, or exon 70 of the DMD gene or any exon chosen among exons 59 to 79 of the DMD gene. More preferably, the nucleotide sequence to be trans-spliced to the target pre-mRNA of the DMD gene thus comprises at least exon 23, or exon 70 of the DMD gene, or any exon chosen among exons 59 to 79 of the DMD gene. Even more preferably the nucleotide sequence to be trans-spliced to the target pre-mRNA of the DMD gene comprises the cDNA from exon 59 to 79 of DMD human gene. The present invention also concerns a method for correcting at least one genetic mutation present in at least one exon of the DMD gene in a subject in need thereof, comprising administering to said subject the simple trans-splicing molecule of the invention. Preferably, said at least one exon of the DMD gene is exon 23 or exon 70, and the nucleotide sequence to be irans-spliced comprises at least the exon 23 of the DMD gene (that is SEQ ID NO 8 for the mouse gene or SEQ ID NO 60 for the human gene), or the exon 70 of the DMD gene (that is, SEQ ID NO 72 for the human gene, or SEQ ID NO 73 for the murine gene), or the cDNA from exon 59 to exon 79 of the normal DMD human gene (SEQ ID NO 70).

In this case, in other words, the invention is drawn to the simple trans-splicing molecule of the invention, or the recombinant vector comprising it, or the cell comprising them, for their use for correcting at least one genetic mutation present in exon 23 or exon 70 of the normal human DMD gene in a subject in need thereof, wherein the nucleotide sequence to be irans-spliced comprises at least the exon 23 of the normal DMD gene (that is SEQ ID NO 8 for the mouse gene or SEQ ID NO 60 for the human gene), or the exon 70 of the normal DMD gene (that is, SEQ ID NO 72 for the human gene, or SEQ ID NO 73 for the mouse gene), or the cDNA from exon 59 to exon 79 of the normal DMD human gene (SEQ ID NO 70). The present invention also provides pharmaceutical compositions comprising an effective amount of the simple PTM of the invention, 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 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 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, i. e. in the muscles. This may be achieved by, for example, and not by way of limitation, local infusion during surgery, loco-regional infusion under high pressure in a limb where the arterial and venous blood flux is intermittently interrupted by a tourniquet, topical application, e. g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. Other controled release drug delivery systems exist, such as nanoparticles, matrices such as controlled-release polymers, hydrogels. The simple PTM will be administered in amounts which are effective to produce the desired effect in the targeted cell. Effective dosages of the simple 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 severity of the DMD being treated, and can be determined by standard clinical techniques.

The simple PTM can also be delivered to cells or stem cells ex vivo which can, in a second step after ex vivo correction, be transferred as a cell transplant to an individual with the goal of correcting an organ or an individual affected by a genetic disease through cell therapy.

EXAMPLES

1. Double trans-splicing molecule of the invention 1.1. Materials and methods

Plasmids constructions The murine DMD minigene target (3993bp (SEQ ID NO 39)) comprising exons

E22, E23, and E24 and the natural E23 flanking intronic sequences was constructed by PCR amplification from mdx genomic DNA and subcloned in pSMD2 into Kpnl site. An ATG in Kozack ACCACCATGG context and a STOP codon were introduced at both sides of the minigene. The E24 for the TS and EX molecules (1 14bp (SEQ ID NO 10)) was amplified from the DMD minigene. The different domains of the TS and EX molecules detailed in Results section were constructed by PCR and subcloned in pSMD2 into Hind 111 and EcoRI between the CMV promoter and the polyA signal. Antisens sequences AS bind to DMD intron 22: AS1 targets nucleotides -763 to -614 (SEQ ID NO 13); AS2, -463 to - 314 (SEQ ID NO 14); AS3 -159 to -10 (SEQ ID NO 15); AS3bis -159 to +5 (SEQ ID NO 58) (where nucleotide +1 is the first E23 nucleotide). The second antisens domains AS' bind to DMD intron 23: AS4, +1801 to +1950 (SEQ ID NO 16); AS5, +2101 to +2250 (SEQ ID NO 17); AS6, +2401 to +2550 (SEQ ID NO 18); AS7, -5 to +145 (SEQ ID NO 19); AS8, +151 to +300 (SEQ ID NO 20) (where nucleotide +1 is the first nucleotide of intron 23). All expression cassettes are under the control of the strong CMV promoter and a polyA signal and were verified by sequencing.

Cell culture and transfection Mouse embryonic fibroblast NIH3T3 cells were maintained in DMEM

(Invitrogen) supplemented with 10% heat-inactivated FBS (Invitrogen), 100 units/ml penicillin, and 100μg ml streptomycin. For transfections, cells were grown to 70% confluence in 12-well plates and exposed to the DNA Lipofectamine 2000 reagent (Invitrogen) complex for 5h in DMEM before being returned to normal culture medium. Typically, 0^g of DMD minigene and 1 ,5 μg of TS or EX molecules DNA were used in each transfection. Cells were routinely analyzed 72h after transfection.

RT-PCR analysis

Total RNA was isolated from transfected cells by using RNAeasy extraction kit (Qiagen). Reverse transcription was performed on 200ng of RNA by using the Superscript II (Invitrogen) and the reverse primer pSMD2-R1 (see below) at 10 min at 25°C, 50 min at 42°C, and a final step of 5 min at 95°C. To detect non-repaired and repaired DMD transcripts, reverse transcribed RNA was amplified by PCR under the following conditions: 95°C for 5 min, 30 cycles of 30 s at 95°C, 1 min at 56°C, 45 s to 1 min at 72°C, and a final step of 7 min at 72°C. The sequences of the primers were as followed: E22-F

G AC ACTTTAC C AC C AATG C G C (SEQ ID NO 36) (Primer A on Fig. 1A-B and 2A-B), pSMD2-R1 CTTTCTGATAGGCAGCCTGC (SEQ ID NO 37) (Primer B on Fig. 1A-B) and pSMD2-R5 CTCACCCTGAAGTTCTCAGG (SEQ ID NO 38) (Primer C on Fig. 2A- B). RT-PCR products were separated by electrophoresis in 2% agarose gels with ethidium bromide and sequenced.

Quantitative real-time RT-PCR mRNA levels were measured by absolute quantitative real-time RT-PCR method using Absolute SYBR Green Rox Mix (Thermo scientific). Two positive control DMD cDNA fragments, E22-E23-E24 and E22-E24-E24, were cloned into the pCR®2.1 -TOPO®. As a reference samples, those plasmids were 10-fold serially diluted (from 10 ⁷ to 10 ³ copies) and used to generate standard curves. Real-time PCR was performed and analyzed on a DNA Engine Opticon 2 (Bio-Rad). In each experiment, duplicates of standard dilution series of control plasmids and first strand cDNA generated by the Superscript II (Invitrogen) from 200ng of total RNA were amplified by specific primers. Primers for E23, E23-F AG ATG G C C AAG AAAG C AC C (SEQ ID NO: 32) and E23-R CTTTCCACCAACTGGGAGG, (SEQ ID NO: 33) were used to measure non-repaired DMD transcript; and primers for E24-E24 junction, E24- F TGAAAAAACAGCTCAAACAATGC (SEQ ID NO: 34) and E24-R AGCATCCCCCAGGGCAGGC (SEQ ID NO: 35), for the repaired transcript. 1. 2. Results

Design of trans-splicing molecules (TS molecules)

In Exchange molecules, the replacing exon is flanked by artificial intronic sequences with strong acceptor and donor splice sites, which are connected to antisense sequences designed to anneal the target mRNA. Annealing is crucial to permit the trans-splicing reaction, although it is not enough. Ideally, the site of annealing must disturb the definition of the targeted exon in the parental pre- messenger while enhancing cross-splicing in between the two independent mRNAs. In the case of Exchange, there are more constraints since two trans-splicing reactions must be synchronized at both edges of the targeted exon. The murine model for DMD, the mdx mouse, carries a nonsense mutation in exon 23 (E23 ^m : SEQ ID NO 9) of the dystrophin gene. In order to locate the best site of annealing in intron 22, upstream the mutated exon, three trans-splicing (TS) molecules for 3' replacement only differing in their binding domains were designed (Fig. 1A). Antisense sequences of about 150 nucleotides (AS1 = SEQ ID NO 13, AS2 = SEQ ID NO 14 and AS 3 = SEQ ID NO 15) were chosen to match either to the 5' end, the middle or the 3' end of intron 22 (SEQ ID NO 1 1 ). The idea was to test whether getting the TS molecule close to its target, 5' donor splice site of intron 22, or at the opposite, masking the 3' acceptor splice site, would facilitate trans-splicing. In the three constructions, the artificial intron included a spacer sequence (SEQ ID NO 59), a strong conserved yeast branch point sequence (SEQ ID NO 25), a polypyrimidine tract (SEQ ID NO 28), and a canonical 3' acceptor splice site (SEQ ID NO 29). To facilitate the readout, it was also decided to employ exon 24 (E24 (SEQ ID NO 10)) in the TS molecule instead of the normal version of exon 23 (E23). Indeed, E24 is smaller than E23 (1 14 versus 213bp) allowing unequivocal distinction by RT-PCR of repaired mRNA (E22-E24-E24) from non-repaired parental transcripts (E22-E23 ^m -E24). Three TS molecules for 3' replacement were thus constructed: AS1 -E24 (SEQ ID NO 40), AS2- E24 (SEQ ID NO 41 ), AS3-E24 (SEQ ID NO 42). As control, a trans-splicing molecule with no binding domain (AS-, SEQ ID NO 43) was used.

To facilitate the analysis of DMD splicing in tissue culture, a DMD reporter gene made of a genomic fragment of 3993bp comprising E22 to E24 with full-length natural introns was made (SEQ ID NO 39). Cis- and trans-splicing patterns are illustrated in Figure 1 B. An RT-PCR strategy was designed to detect specifically RNA resulting from cis- and trans-splicing events by using a forward primer E22-F (SEQ ID NO 36) (arrow A in Fig. 1 B) specific for E22, and a reverse primer pSMD2-R1 (SEQ ID NO 37) (arrow B) specific for a sequence upstream the polyA signal in DMD minigene and TS molecules. Importantly, these primers also allowed discriminating E22-E24 amplicons resulting from either trans-splicing or exon skipping.

3' replacement in DMD transcripts

DMD reporter minigene and TS plasmids were cotransfected in the mouse embryonic fibroblast NIH3T3 cell-line. Cells were harvested 72h after transfection, and total RNA was isolated. Cis- and trans-spliced RNA patterns were assessed by RT- PCR. As expected, samples that received only the DMD minigene displayed a single 638bp amplicon corresponding to the cis-spliced DMD transcript E22-E23 ^m -E24 (Ctrl in Fig. 1 C). Also, cDNAs from cells transfected with both DMD minigene and trans- splicing constructs (AS2-E24) gave no PCR products when reverse transcription was omitted (RT- AS2), ensuring about the specificity of the present assay. In the presence of U7-SD23-BP22 (U7) plasmids described to induce E23 skipping (Goyenvalle et al., 2004), a 425bp band corresponding to E22-E24 transcript from cis-splicing was detected.

In samples that received DMD minigene and TS plasmids, a product of 310bp was generated, corresponding specifically to the trans-spliced E22-E24 variant, and not to an exon skipping product as it was obtained with U7. In the presence of either AS1 - E24 (SEQ ID NO 40) or AS2-E24 (SEQ ID NO 41 ), the E22-E23 ^m -E24 amplicon corresponding to the parental DMD minigene had almost entirely disappeared thus confirming that, here, trans-splicing efficacies were nearly complete. Importantly, trans- splicing did not occur when AS was removed (AS-) from TS constructs demonstrating that this reaction required close interaction in between the two strands of mRNA to combine. The AS3-E24 TS molecule (SEQ ID NO: 42) appeared to be less efficient. Surprisingly, extending AS3 in order to cover the 3' acceptor site of E23 ^m did not improve the trans-splicing reaction (not shown). These experiments show that trans- splicing could not do without AS sequences, although getting closer the two mRNAs is not sufficient. mRNA repair by using Exchange

To test the possibility of mRNA repair by Exchange, several Exchange (EX) molecules AS-E24-AS' based on the efficient TS molecules mentioned above were developed, and modified to bind both intron 22 and intron 23 of the DMD reporter minigene (Fig. 2A). The EX molecules contained the same elements as previously described in AS1 -E24 and AS2-E24 TS molecules followed by a 5' donor splice site (SEQ ID NO 30) and a second 150nt antisense targeting intron 23 (SEQ ID NO 12). Five antisenses, AS 4 (SEQ ID NO 16), AS5 (SEQ ID NO 17), AS6 (SEQ ID NO 18), AS7 (SEQ ID NO 19) and AS8 (SEQ ID NO 20), were selected within intron 23 (SEQ ID NO 12), which spans over 2607bp. The following spacer sequences were used: for the spacer sequence that separates the 3' splice region from the 5'-end target binding domain AS, spacer 2 (SEQ ID NO 24, 42 nucleotides) was used, and for the spacer sequence that separates the 5' splice region from the 3'-end target binding domain AS', spacer 1 (SEQ ID NO 23, 34 nucleotides) was used.

Finally, the following PTM were constructed: AS1 -E24-AS4 (SEQ ID NO 44), AS1 -E24-AS5 (SEQ ID NO 45), AS1 -E24-AS6 (SEQ ID NO 46), AS1 -E24-AS7 (SEQ ID NO 47), AS2-E24-AS4 (SEQ ID NO 48), AS2-E24-AS5 (SEQ ID NO 49), AS2-E24- AS6 (SEQ ID NO 50), AS2-E24-AS7 (SEQ ID NO 51 ), AS2-E24-AS8 (SEQ ID NO 52) and AS2-E24-2XAS4 (SEQ ID NO 53).

As previously, EX constructs and DMD minigene were cotransfected in NIH3T3 cell-line. Cells were harvested 72h after transfection, and total RNA was extracted. To detect specifically RNA resulting from cis-splicing and/or exon exchange events, a forward primer E22-F (SEQ ID NO 36) (arrow A in Fig. 2A-B) specific of E22, and a reverse primer pSMD2-R5 (SEQ ID NO 38) (arrow C) specific of a sequence only present in the DMD minigene upstream its polyA signal were used. Targeting of AS- E24-AS' in the DMD reporter pre-mRNA is illustrated in Figure 2A and expected sizes of the various amplification products shown in Figure 2B.

A RT-PCR product of 408bp was detected in samples transfected with AS2- E24-AS' plasmids (Fig. 2C). Direct sequencing confirmed that this product corresponded to the exchanged mRNA variant E22-E24-E24 (Fig. 2D). This product was absent when one of the two antisens was lacking, showing that co-targeting of intron 22 and intron 23 is crucial for Exchange. Among the antisense combinations we tried, levels of the 408bp band were stronger with AS2-E24-2XAS4 (SEQ ID NO 53), - AS7 (SEQ ID NO 51 ) and -AS8 (SEQ ID NO 52). Interestingly, the AS2-E24-2XAS4 molecule (SEQ ID NO 53), which carried two AS4, was more efficient than its single AS4 counterpart. In AS2-E24-AS7 sample, a supplementary band of 294bp was detected corresponding to E22-E24 transcript generated by exon 23 skipping. This was not surprising considering that AS7 bound the 5' donor splice site of intron 23 and would mask its recognition by the spliceosome. It is likely that AS4, AS7 and AS8 brought back EX molecules closer to E23 than AS5 and AS6 suggesting that a tight framing is essential for efficient Exchange.

Optimization of Exchange efficacy by adding up intronic splice enhancers

In order to improve the Exchange reaction, the G-rich intronic splice enhancer (ISE, SEQ ID NO 26) from the human GH-1 gene was added upstream the 3' acceptor site of AS2-E24-AS4 and AS2-E24-AS8 (McCarthy & Phillips, 1998) and the DISE sequence from the rat FGFR2 gene downstream the 5' donor site (SEQ ID NO 27) (Kierlin-Duncan & Sullenger, 2007) (Fig. 3A). As shown in Figure 3B, RT-PCR analysis revealed that insertion of the DISE sequence in AS2-E24-AS4 (SEQ ID NO 54) and AS2-E24-AS8 (SEQ ID NO 55) molecules increased significantly the 408bp band corresponding to the E22-E24-E24 mRNA variant, while addition of ISE sequence did not enhance the Exchange efficacy. As expected, no Exchange was observed with control vectors lacking the downstream AS': AS2-ISE-E24 and AS2-E24-DISE. Figure 3C shows Exchange efficacy of various vectors by using quantitative RT-PCR. The AS2-DISE-E24-AS4 (SEQ ID NO 54) molecule allowed obtaining 53% of exon exchange. Its efficacy was improved by about 7,5 folds when compared to its counterpart AS2-E24-AS4 (SEQ ID NO 48) lacking the DISE motive. Interestingly, introduction of two redundant downstream AS' (here AS4) improved Exchange efficacy, which was about 30%. However, AS2-E24-DISE-2XAS4 (SEQ ID NO 57) was not more efficient than AS2-DISE-E24-AS4 (SEQ ID NO 54)(data not shown).

2. Simple trans-splicing molecule of the invention 2.1. Materials and Methods

Plasmid constructions

The different domains of the TSM molecules were constructed by PCR and subcloned into pSMD2. Human dystrophin exons 59 to 79 until the STOP codon (2390bp, see SEQ ID NO 70) were amplified from human myotubes cDNA while antisense sequences from human genomic DNA. Antisense sequence binds to dystrophin intron 58: nucleotides -445 to -295 (where nucleotide +1 is the first E59 nucleotide, SEQ ID NO 68). The trans-splicing cassettes were subcloned into a plasmid pRRL-cPPT-mcs-WPRE under hPGK promoter (Zufferey R et al, 1997). All the constructs were verified by sequencing.

Sequences

Artificial intron included a spacer sequence, a strong conserved yeast branch point sequence (BP), a polypyrimidine tract (PPT) and a canonical 3' splice acceptor site (SA): the same sequences are already described for the double PTM of example 1 .

Cell cultures

Primary DMD myoblast cell cultures were established from explants of biceps as described previously (Mouly V et al, 1993), in accordance with French ethics legislation. DMD and control myoblasts CHQ (Edom et al, 1994) were grown in Skeletal Muscle Cell Growth Medium (PromoCell). To induce differentiation, cultures were switched to DMEM 2% horse serum with apotransferrin (100μg ml) and insulin (10μg ml). All cultures were grown in humidified incubators at 37°C in 5% C02. Lentiviral productions

Lentiviral vectors pseudotyped with the VSV-G protein were produced by transient quadri-transfection into 293T cells were determined by transduction of HCT1 16 cells and assayed by quantitative real-time PCR on genomic DNA [Charrier S et al, 2005). Titration of the lentivirus is expressed as viral genome/mL (vg/mL) (ranging from 2.5X10 ⁹ to 5X10 ⁹ vg/mL). 5X10 ⁶ vg were used to transduce 4X10 ⁵ myoblasts plated the day before in 24 wells tissue culture dishes in 500 μΙ_ of DMEM supplemented with 10% FCS. Four hours post-transduction, the medium was diluted by adding 200 μΙ_ per well of previous medium. The dishes were incubated for 24 hr at 37°C and 5% C02 before washing.

RT-PCR analysis

Total RNA was extracted from transduced myotubes with TRIzol reagent (Invitrogen). Five microgram of RNA was reverse transcribed using Superscript III First-Strand Synthesis SuperMix (Invitrogen) and the reverse primer Pst1 -WPRE-Ro, AACTGCAGCAGGCGGGGAGGCGGCCCAAAG (Ro on Fig. 2A). cDNAs were subjected to nested PCR amplification with Phusion High-Fidelity PCR Master Mix with GC buffer (Finnzymes) under the following conditions: 95°C for 5 min, 20 cycles of 30 s at 95°C, 2 min at 56°C, 45 s to 1 min at 72°C, and a final step of 7 min at 72°C, using external primers E58-Fo CATGAGTACTCTTGAGACTG (Fo on Fig. 2A) and WPRE-Ro AGCAGCGTATCCACATAGCG (Ro). Five microliters of each of these reactions was then reamplified for 30 cycles using the internal primers E58-Fi, AGGACTAGAGAAACTCTACC (Fi), and WPRE-Ri, TTGTCGACCAGCGTTTCTAG (Ri). RT-PCR products were separated by electrophoresis in 1 % agarose gels with ethidium bromide and sequenced. Protein analysis

Forty μg of protein were loaded onto NuPAGE® Novex 4-12% Bis-Tris Gel (Invitrogen), electrophoresed, blotted onto nitrocellulose membranes and probed with 1 :50 NCL-DYS1 or NCL-DYS2 (NovoCastra), followed by incubation with 1 :15000 horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch) and SuperSignal® West Pico Analysis System (Thermo Scientific). 2.2. Results

The aim of this work was to repair by simple trans-splicing dystrophin pre- mRNAs carrying any genetic anomalies present between exons 59 to 79, which represents 8% of Duchenne patients. These patients are not treatable by exon skipping therapy as these exons are indispensable for the protein function.

The simple trans-splicing molecule contains a 150 nucleotides antisense sequence complementary to intron 58 (SEQ ID NO 68), an artificial intron included a spacer sequence (SEQ ID NO 23), a strong conserved yeast branch point sequence (BP) (SEQ ID NO 25), a polypyrimidine tract (PPT) (SEQ ID NO 28) and a canonical 3' splice acceptor site (SA) (SEQ ID NO 29), and the normal human dystrophin cDNA from exon 59 to the exon 79 STOP codon (SEQ ID NO 70) (cf. figure 4). A RT-PCR strategy was designed to amplify specifically a 2443bp product from mRNA resulting from trans-splicing events by using forward primers, E58-Fo and E58-Fi (arrows Fo/Fi in figure 5A), specific for E58, and reverse primers, WPRE-Ro and WPRE-Ri (Ro/Ri in figure. 5A), specific for the WPRE element present on trans-splicing molecules.

Duchenne muscular dystrophy patient myoblasts carrying a non-sense mutation in exon 70 were transduced with lentivirus expressing trans-splicing molecules. After differentiation, myotubes were harvested, total RNA isolated and trans-spliced dystrophin transcripts investigated by specific RT-PCR. As expected, samples that did not receive lentivirus gave no PCR products (lane "-" in figure 5B). In samples that express trans-splicing molecules (TsM), a product of 2443bp was generated corresponding specifically to the trans-spliced DMD variant as confirmed by direct sequencing of the amplicon (data not shown).

In order to detect specifically rescued dystrophin encoded by trans-spliced transcripts, Western blotting with the NCL-DYS1 monoclonal antibody that recognizes spectrin-like repeats R8 to R10 was used. Consistent with the generation of trans- spliced transcripts, the full-length 427kD dystrophin protein was readily detected by Western blot on transduced myotubes extracts (the two lanes TsM in figure 6), whereas no band is present in the non treated DMD cells ("-"). BIBLIOGRAPHIC REFERENCES

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