WO2002066076A1

WO2002066076A1 - A method of treatment and agents useful for same

Info

Publication number: WO2002066076A1
Application number: PCT/AU2002/000169
Authority: WO
Inventors: Robert Kapsa; Andrew Kornberg
Original assignee: Melbourne Neuromuscular Research Institute
Priority date: 2001-02-21
Filing date: 2002-02-18
Publication date: 2002-08-29

Abstract

The present invention relates generally to a method for the treatment and/or prophylaxis of a disorder characterised by the expression of one or more defective genes and to agents useful for same. More particularly, the present invention is directed to a method for the treatment and/or prophylaxis of a disorder characterised by the expression of one or more defective genes by genetically repairing autologous defective cells in order to facilitate the expression of a non-defective form of the subject gene. The method of the present invention is useful in a range of disease conditions such as, but not limited to neuromuscular disease of genetic origin, motor neurone disease, triplet repeat disorders, myotonic dystrophy, fascio-scapular humeral dystrophy, other muscular dystrophies involving gene mutation and conditions characterised by dystrophin deficiency such as muscular dystrophy and still more particularly Duchenne Muscular Dystrophy and Becker's Muscular Dystrophy.

Description

A METHOD OF TREATMENT AND AGENTS USEFUL FOR SAME

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

Bibliographic details of the publications referred to by author in this specification are collected alphabetically at the end of the description.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

The most common of the muscular dystrophies, Duchenne Muscular Dystrophy (DMD), is an X-linked recessive disorder for which no effective therapy is currently available and which confers significant personal, social, emotional and financial hardships to families with affected boys. The condition affects 1 in 3,300 boys, diagnosed at 4 to 5 years of age, usually because of gait abnormalities and approximately 1/3 of cases arise by spontaneous mutation. By early teens, boys are wheelchair bound and continued steady progression of clinical symptoms and ongoing muscle degeneration leads to death in the early 20's as a result of respiratory or cardiac failure. DMD is characterised by structural deficiencies of a protein called dystrophin. Dystrophin is a large 427 kDa rod-like cytoskeletal protein composed of 3, 685 amino acids, found predominantly in muscle fibres, with some incidence in brain and other cells. The human protein shows -90% homology with its murine counterpart and is divided into 4 distinct domains: The N-terminal (240 amino acids) is followed by a helical rod domain (2,400 aa), a Cysteine-rich region (280 aa) and the C-terminal domain (420 aa). Normally, dystrophin performs a crucial structural role in maintenance of myofibre stability and integrity in myogenic cells (Hoffman et ah, 1988) and in other cell types appears to have a similar structural function. In particular, the Cys- rich region encompassing aa3080 to aa3360 in addition to the C-terminus of the mature peptide forms links with the extra-cellular matrix via β-Dystroglycan and Syntrophin respectively (Ambrose et al, 1997), with both regions binding dystrophin-associated glycoproteins (DAGs). The N-terminal of the peptide completes this link by association with Actin in myogenic cells. Shorter dystrophin isoforms found normally in tissues of non-muscle origin, range in size from 71 kDa (Glial cells) to 260 kDa (Retinal epithelium), some of which are exclusive of the N-terminal regions. In all isoforms of normally expressed dystrophin, the Cys-rich and C-terminal regions are present. The dystrophin peptide from boys with DMD is a severely truncated form of the normal peptide, in which the peptide's Cys-rich region and/or C-terminal (C-terminal domains) are missing or disrupted. In DMD these C-terminal truncations lead to severe degeneration of muscle. In contrast, Becker's Muscular Dystrophy (BMD) also involves large truncations of the dystrophin protein generally in the helical rod domain, but with overall preservation of the protein's C-terminal regions, results in a considerably milder condition.

The dystrophin gene (dys) contains 79 exons distributed throughout 2,500,000 base pairs of chromosome Xp21.2. The introns occupy 99.4% of the gene sequence with the fully processed transcript being only 14kp. The critical Cys-rich and Syntrophin binding domains towards the C-terminal of dystrophin are encoded on exons 62 to 72 and exon 74 of the dys gene respectively. Expression of the gene is complex and occurs in cell-specific and developmentally regulated manner with at least 7 independent promoters known throughout the gene (Nobile et al., 1997). In DMD 96% of cases involve frameshift or nonsense mutation of the dystrophin gene, with the remainder involving in-frame deletions of the Cys-rich and C-terminal domains. These dys gene mutations result in severe disruption of the critical β-Dystroglycan (Cys-rich), DAG and Syntrophin (C-terminal) binding regions of the mature dystrophin peptide, which in turn underlies the instability of the myofϊbre plasma membrane characteristic to DMD.

In 65% of Duchenne boys, the gene contains gene rearrangements (predominantly frameshift deletions or duplications), whilst the remaining 35% have a dys gene with either nonsense mutations or mutations that affect transcriptional splicing sites. A very small proportion of DMD individuals have in-frame dys rearrangements which involve the two critical C-terminal binding domains.

In the milder BMD, 85% of cases are characterised by deletions, with the remainder being a variety of point mutations. Becker dys locus mutations mostly preserve the C-terminal reading frame with most affecting the helical rod domain via in-frame deletions of missense point mutations. This translates to a far milder dystrophic condition mediated by preservation of the dystrophin C-terminal.

Currently the only treatment available to boys with DMD involves Steroid drugs such as Prednisone, Deflazacort or Oxandrolone which delay the disease progression for 2 to 3 years. In addition, at several points throughout their lives, Duchenne boys undergo major corrective surgery to deal with various effects such as Scoliosis and others, all resulting from progressive muscle failure. Considerable effort has been extended by groups worldwide including over the past decade to develop strategies to provide myogenic cells in boys with DMD with normal dystrophin or to otherwise fortify the dystrophic muscle. These include viral gene therapy, cytokine stimulation of myogenic precursor proliferation, up-regulation of the dystrophin homologue utrophin, relaxation of codon recognition by aminoglycoside antibiotics, ribo-oligo induced exon skipping, Myoblast Transplantation Therapy (MTT) and Bone Marrow Stem cell transplantation (BMSCT). It has been shown that infusion of Leukemia Inhibitory Factor (LIF) into damaged muscle in vivo results in significant enhancement of muscle regeneration (Barnard et al., 1994; Kurek et al., 1996). In more recent work a reversal of the dystrophic process in max mouse diaphragm after administration of LIF using a sustained release alginate rod delivery system has been shown (Austin et al., 1997; 2000). Further, it has been shown that MTT efficiency can be improved by up to 500% with sustained LIF release during MTT (Bower et al., 1997; Fig 3). Although cytokines can improve MTT graft success, there still remains the most significant obstacle to MTT, and indeed to all cell transplantation/viral therapies, of donor cell rejection by the recipient's immune system.

Accordingly, there is a need to develop methods of treating muscular dystrophy which overcome these histocompatability issues. In work leading up to the present invention, the inventors have developed a method of increasing functional dystrophin levels in an individual exhibiting a defective dystrophin gene by genetically repairing autologous defective cells in order to facilitate their production of functional dystrophin. By repairing autologous cells, the problems associated with foreign tissue rejection are overcome. Accordingly, this work has thereby facilitated the development of a method of treating any condition which is characterised by the expression of a defective gene based on genetically repairing an effective number of autologous cells, either in vitro or in vivo, thereby restoring an affected individual's ability to express a non-defective form of the subject gene without the concomitant complication of histocompatability problems which are usually associated with the introduction of foreign cells or tissue to an individual.

SUMMARY OF 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 subject specification contains nucleotide and amino acid sequence information prepared using the programme Patentln Version 3.1, presented herein after the bibliography. Each nucleotide sequence is identified in the sequence listing by the numeric indicator <201> followed by the sequence identifier (eg. <210>1, <210>2, etc). The length, type of sequence (DNA, protein (PRT), etc) and source organism for each nucleotide sequence is indicated by information provided in the numeric indicator fields <211>, <212> and <213>, respectively. Nucleotide sequences referred to in the specification are defined by the information provided in numeric indicator field <400> followed by the sequence identifier (eg. <400>1, <400>2, etc).

One aspect of the present invention is directed to a method for treating a condition in an individual, which condition is attributable at least in part to the expression of a defective gene, said method comprising administering to said individual an effective number of autologous genetically altered cells, wherein said cells in their non-altered form are characterised by the subject defective gene, said genetic alteration comprising the introduction of at least one modification to said defective gene wherein said genetically altered cells are characterised by the capacity to produce an expression product or derivative, homologue, analogue, mutant or mimetic thereof which exhibits improved functional activity relative to the expression product produced by said defective gene.

Another aspect of the present invention is directed to a method for treating dystrophin deficiency in an individual, which deficiency is attributable at least in part to defective dystrophin production, said method comprising administering to said individual an effective number of autologous genetically altered cells, which cells in their non-altered form are characterised by a defective dystrophin gene, said genetic alteration comprising the introduction of at least one modification to the dystrophin encoding gene wherein said genetically altered cells are characterised by the capacity to produce dystrophin or derivative, homologue, analogue, mutant or mimetic thereof, which dystrophin exhibits improved functional activity relative to the expression product produced by said defective dystrophin gene.

Yet another aspect of the present invention, therefore, there is provided a method for treating dystrophin deficiency in an individual, which deficiency is attributable at least in part to defective dystrophin production, said method comprising administering to said individual an effective number of genetically altered autologous myoblasts, which myoblasts in their non-altered form are characterised by a defective dystrophin gene, said genetic alteration comprising the introduction of at least one modification to the dystrophin encoding gene wherein said genetically altered myoblasts are characterised by the capacity to produce dystrophin or derivative, homologue, analogue, mutant or mimetic thereof, which dystrophin exhibits improved functional activity relative to the expression product produced by said defective dystrophin gene.

In still another aspect there is provided a method for treating dystrophin deficiency in an individual, which deficiency is attributable at least in part to defective dystrophin production, said method comprising administering to said individual an effective number of genetically altered autologous stem cells, which stem cells in their non-altered form are characterised by a defective dystrophin gene, said genetic alteration comprising the introduction of at least one modification to the dystrophin encoding gene wherein said genetically altered stem cells are characterised by the capacity to produce dystrophin or derivative, homologue, analogue, mutant or mimetic thereof, which dystrophin exhibits improved functional activity relative to the expression product produced by said defective dystrophin gene.

In still yet another aspect there is provided a method for treating dystrophin deficiency in an individual, which deficiency is attributable at least in part to defective dystrophin production, said method comprising administering to said individual an effective number of genetically altered autologous cells, which cells in their non-altered form are characterised by a frame shift, nonsense mutation or in frame deletion of the dystrophin gene region encoding the cysteine-rich and/or C-terminal domains of dystrophin, said genetic alteration comprising the introduction of at least one modification to said dystrophin encoding gene wherein said genetically altered cells are characterised by the capacity to produce dystrophin or derivative, homologue, analogue, mutant or mimetic thereof, which dystrophin exhibits improved functional activity relative to the expression product produced by said defective dystrophin gene.

In yet still another aspect there is provided a method for treating dystrophin deficiency in an individual, which deficiency is attributable at least in part to defective dystrophin production, said method comprising administering to said individual an effective number of genetically altered autologous cells, which cells in their non-altered form are characterised by a frame shift, nonsense mutation or in-frame deletion of the dystrophin gene region encoding the cysteine-rich and/or C-terminal domains of dystrophin, said genetically alteration comprising the introduction of at least one modification to one or more of exons 62-72 or 74 of said dystrophin encoding gene wherein said genetically altered cells are characterised by the capacity to produce dystrophin or derivative, homologue, analogue, mutant or mimetic thereof, which dystrophin exhibits improved functional activity relative to the expression product produced by said defective dystrophin gene.

A further aspect of the present invention provides a method of treating dystrophin deficiency in an individual, which deficiency is attributable at least in part to defective dystrophin production, said method comprising administering to said individual an effective number of genetically altered autologous cells, which cells in their non-altered form are characterised by a nucleotide deletion or point mutation of the dystrophin gene region encoding the helical rod domain of dystrophin, said genetic alteration comprising the introduction of at least one modification to said dystrophin encoding gene wherein said genetically altered cells are characterised by the capacity to produce dystrophin or derivative, homologue, analogue, mutant or mimetic thereof, which dystrophin exhibits improved functional activity relative to the expression product produced by said defective dystrophin gene.

Another further aspect of the present invention provides a method for treating a condition in an individual, which condition is attributable at least in part to the expression of a defective gene, said method comprising administering to said individual an effective number of autologous genetically altered cells, which cells in their non-altered form are characterised by the subject defective gene, said genetic alteration comprising the introduction of a nucleic acid molecule or derivative or analogue thereof to the subject gene by small fragment homologous recombination, wherein said genetically altered cells are characterised by the capacity to produce an expression product or derivative, homologue, analogue mutant or mimetic thereof which exhibits improved functional activity relative to the expression product produced by said defective gene.

In still another further aspect there is provided a method for treating dystrophin deficiency in an individual, which deficiency is attributable at least in part to defective dystrophin production, said method comprising administering to said individual an effective number of genetically altered autologous cells, which cells in their non altered form are characterised by a defective dystrophin gene, said genetic alteration comprising the introduction of a nucleic acid molecule or derivative or analogue thereof to the dystrophin encoding gene by small fragment homologous recombination, wherein said genetically altered cells are characterised by the capacity to produce dystrophin or derivative, homologue, analogue, mutant or mimetic thereof, which dystrophin exhibits improved functional activity relative to the expression product produced by said defective dystrophin gene.

Yet another aspect of the present invention is directed to the therapeutic or/or prophylactic treatment of a disease condition, which disease condition is characterised by the expression of a defective gene, said method comprising administering to said individual an effective number of genetically altered autologous cells, which cells in their non-altered form are characterised by the subject defective gene, said genetic alteration comprising the introduction of at least one modification to said defective gene wherein said genetically altered cells are characterised by the capacity to produce an expression product or derivative, homologue, analogue, mutant or mimetic thereof which exhibits improved functional activity relative to the expression product produced by said defective gene.

Still yet another aspect of the present invention is directed to the therapeutic and/or prophylactic treatment of a disease condition, which disease condition is characterised by a dystrophin deficiency attributable at least in part to defective dystrophin production, said method comprising administering to said individual an effective number of genetically altered autologous cells, which cells in their non-altered form are characterised by a defective dystrophin gene, said genetic alteration comprising the introduction of at least one modification to the dystrophin encoding gene wherein said genetically altered cells are characterised by the capacity to produce dystrophin or derivative, homologue, analogue, mutant or mimetic thereof, which dystrophin exhibits improved functional activity relative to the expression product produced by said defective dystrophin gene.

In yet still another further aspect there is provided a method for the therapeutic and/or prophylactic treatment of Duchenne Muscular Dystrophy said method comprising administering to said individual an effective number of autologous genetically altered cells, which cells in their non-altered form are characterised by a defective dystrophin gene, said genetic alteration comprising the introduction of at least one modification to the dystrophin encoding gene wherein said genetically altered cells are characterised by the capacity to produce dystrophin or derivative, homologue, analogue, mutant or mimetic thereof, which dystrophin exhibits improved functional activity relative to the expression product produced by said defective dystrophin gene.

Another aspect of the present invention provides a method for the therapeutic and/or prophylactic treatment of Becker's Muscular Dystrophy, said method comprising administering to said individual an effective number of autologous genetically altered cells, which cells in their non-altered form are characterised by a defective dystrophin gene, said genetic alteration comprising the introduction of at least one modification to the dystrophin encoding gene wherein said genetically altered cells are characterised by the capacity to produce dystrophin or derivative, homologue, analogue, mutant or mimetic thereof, which dystrophin exhibits improved functional activity relative to the expression product produced by said defective dystrophin gene.

Still another aspect of the present invention relates to the use of genetically altered cells, as hereinbefore defined, in the manufacture of a medicament for the treatment of a condition characterised by the expression of a defective gene.

Still yet another aspect of the present invention relates to genetically altered cells which produce an expression product or a derivative, homologue, analogue, mutant or mimetic thereof, as hereinbefore defined, which exhibits improved functional activity relative to the expression product produced by the non-altered cells.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic representation of the dys Gene SFHR Strategy in the mdx Mouse.

Figure 2 is an image of Allele-Specific PCR Dection of wt dys Locus.

Figure 3 is a graphical representation of a 500% improvement of MTT with LIF in mdx mice.

Figure 4 is an image of a 100%o wt dys locus in mdx Bone Marrow.

Figure 5 is an image of SFHR I: Repair of the mdx dys locus.

Figure 6 is an image of Quantification of SFHR Efficiency.

Figure 7 is an image of SFHR II: Multiple transfections.

Figure 8 is an image of SFHR in vivo (AS-PCR).

Figure 9 is a schematic representation of the strategy for dys_. Gene Repair by SFHR. Sequence data was obtained for 876 bp of murine dys gene regions including partial sequence of introns 22 and 23 and the entire exon 23 sequence. Oligonucleotide primers A, B, C (sense) and D (antisense) were designed to optimize access to the known sequence at this locus. The oligonucleotide primers were mapped according to this known sequence as shown in Table 1. A 603 bp section of this region was amplified from wt C57BL/10 mouse DNA by PCR with primers C and D to generate Amplicon C which contained the wt nucleotide (C) at the mdx locus. Amplicon C was then used to transfect mdx myoblasts and induce homologous replacement of the mutant nucleotide (T) with the wt C. Any subsequent PCR amplification using primers A and B of DNA extracted from the transfected mdx cultures generated an amplicon that could only originate from the chromosomal locus. Figure 10 is an image of the detection and quantification of wt Nucleotide at the dys Locus. A). PCR primer Dys wt AS-01 (Table 1) has a 3' mismatch with the mutant T nucleotide at the mdx locus. PCR amplification using primers B and Dys wt AS-01 was performed on DNA extracted from skeletal muscle of a 9 day old mdx mouse (from which myoblast cultures were also made, see Fig. 3) and from the wt female mouse from which the wt Amplicon C and wt control cultures (Fig. 2A) used in this study were generated. By variation of the annealing temperature during PCR, a 415 bp product can be amplified only from wt DNA. The use of primer B in this assay ensures that only chromosomal locus is amplified. The strategy for PCR-RFLP quantification of repaired cells is shown in B). PCR was performed using primers A and D to amplify 810 bp (Amplicon A) across the repaired region of the dys locus in DNA extracted from myoblasts subjected to SFHR. After purification, Amplicon A was used as template for a second PCR using primers Dys Mae S-01 and Dys Ex23 AS-01, the 104 bp product of which was digested using Mae III. Primer Dys Mae S-01 is modified near the 3' end such that a Mae III site is introduced in the presence of wt DNA immediately downstream of the primer annealing site (Table 1). This results in Mae III site acquisition in the 44 bp fragment (B: Basis) to generate a doublet band (C: Cut) consisting of bands 24 and 20 nucleotides in length. The digestion products are resolved on a 16% non-denaturing acrylamide gel and visualized by phosphorimagery. Incomplete digestion in wt control DNA using Mae III is shown. The extent of digestion can be estimated by comparison of relative intensities of bands S (60 bp; Standard) and B (Basis) in 100% wt control DNA. Amount of wt nucleotide is estimated by comparison of relative intensities of Band B (44 bp) and the C (Cut, 24/20 bp) doublet. The mdxlC control shows that the maximum amount of Amplicon C (10 ng) expected to be co-purified with DNA from SFHR-repaired cultures will not generate an artefactual result during PCR-RFLP detection when added to mdx template prior to amplification of Amplicon A.

Figure 11 is an image of the gene repair at the mdx locus by SFHR. Myoblasts were cultured from a 9 day old mdx male (Culture A) and a 4 mth old wt female (Culture B) and DNA extracted from 10⁵ cells. AUele-specific PCR confirmed the genotype of these myoblasts (Controls A and B, also in Fig 2). Controls Al, X and C were; culture A DNA mixed with 10 ng Amplicon C (prior to PCR), reagent blank (no template DNA) and Amplicon C alone respectively. The 10 ng of Amplicon C in the Al control is the maximum ratio of Amplicon C and mdx culture A DNA that would be present if all of the Amplicon C added during SFHR of culture A was to co-purify with the repaired cultures' (la, lb and lc), DNA. DNA extracted from culture A following one (culture la), two (culture lb), and three (culture lc) cycles of SFHR (using Lipofectamine/Plus) showed the presence of wt C at the mdx locus of the dys gene. SFHR efficiency is visibly improved by SFHR applications subsequent to the first. Cultures 2 and 3 were independent myoblast cultures established from the 9-day old mdx male mouse and repaired once by SFHR. Culture 4 is a totally independent culture (different mouse) that had been subjected to SFHR once 28 days prior to DNA extraction. Numbers in brackets reflect days after SFHR prior to DNA extraction.

Figure 12 is an image of SFHR II: Double application and Lipofection variation. Myoblast cultures were generated from a 4-mth old mdx mouse, which were then divided into 5 tandem cultures (mdx, X, la, 3 and 3a). SFHR was performed on cultures 1 and la once using Lipofectamine (no Plus reagent) and on cultures 3 and 3 a once using Lipofectin. Culture mdx was grown without SFHR. Cultures 1 and la were grown to passage, with one passage from each being grown further and the other being subjected to SFHR one further time to generate cultures 2 and 2a (SFHR=twice). Cultures 3 and 3 a were passaged, with one passage harvested for DNA analysis and the other for myotube differentiation. Cultures 1, la, 2 and 2a were likewise passaged to generate a DNA fraction and a differentiated fraction for dys gene expression analysis. Allele-specific PCR shows that cultures 1 and la had minimal levels of repair after a single cycle of SFHR (Lipofectamine), which was visibly improved by a further cycle (cultures 2 and 2a; 2a as- PCR not shown). Alternatively, as-PCR revealed much more significant levels of gene repair after a single SFHR cycle using Lipofectin (cultures 3 and 3 a). By titration of wt and mdx DNA template and application of PCR-RFLP (lower panel), phosphorimager data verified the semi-quantitative as-PCR result, and showed that 15% to 20%o of dys loci in cultures 2 and 2a and up to 2% of loci in cultures 3 and 3 a had been converted to wt at the mdx position. Cultures 1 and la could not be quantified using PCR-RFLP, which indicates that repair levels may have been as low as 5xl0^"5% of loci in the initial SFHR cycle (data not shown).

Figure 13 is an image of Dystrophin gene expression in repaired cultures. Myoblasts, cultured from a 9-day old male mdx mouse were subjected to two cycles of SFHR (cultures 2 and 2a, Figure 4) SFHR, and a sub-passage allowed to differentiate into myotubes. RNA was extracted from these cultures after 7 days of differentiation, pooled and first-strand RT PCR performed using dys gene-specific (c3603-AS and c2801-S; Table 1) and poly A- specific oligonucleotide primers. The cDNA products were used for amplifying sections of dys (A) and GAP-DH (B) transcripts respectively by PCR. Analysis of dys transcript was performed on equal amounts of template (see methods) generated from the twice- transfected (2&2a) and sham-transfected (transfected without any DNA; mdx-C) myotubes, mdx muscle (mdx-m) and wt muscle (wt-m). After 7 days, neither the transfected (2&2a) or sham-transfected (mdx-C) myotubes expressed dystrophin transcript. An equal amount of mRNA from mdx (mdx-m) and wt

muscle on the other hand, was shown to express detectable dystrophin transcripts by this method. The integrity of mRNA in the transfected cultures (2&2a) was compared to that in the muscle, using GAP-DH expression as a standardizing factor (B). These RT PCR experiments showed that in the presence of equal quantities of starting mRNA, the GAP-DH transcript was equally detectable in the cultured cells as in the muscle. The wt-mxA lane shows the effect of a four-fold increase in wt-m mRNA at the start of the RT.

Figure 14 is an image of Dystrophin Gene Repair by SFHR in mdx Tibialis Anterior. Right Tibialis Anterior (TA) from four 12-week old male mdx mice was injected with SFHR cocktail after bupivacain treatment, whilst Left TA was injected with saline/vehicle. The mice were then killed and both TAs were dissected out, snap-frozen and analyzed for dystrophin expression by anti-dystrophin immunohistochemistry. Following histochemistry, the remaining TA was ground to a powder and the DNA extracted. Allele- Specific PCR was applied to the DNA from both sides and showed evidence of repair in the right TAs compared to the left TAs. Repair was not sufficient to allow evaluation of repaired gene dystrophin expression by RT PCR RFLP. DETAILED DESCRIPTION OF THE INVENTION

The present invention is predicated, in part, on the development of a method of facilitating the expression of a non-defective form of a gene in an individual who expresses a defective from of the subject gene. The method is based on the genetic repair of the subject gene in a number of autologous cells effective to at least partly restore expression of a non- defective form of the gene. By repairing autologous cells, the method of the present invention overcomes the tissue rejection complications inherent in introducing a non-self population of cells which express a non-defective form of the subject gene. The development of this method now facilitates its application in a wide variety of circumstances including, in particular, the therapeutic or prophylactic treatment of disease conditions characterised by the expression of a defective gene.

Accordingly, one aspect of the present invention is directed to a method for treating a condition in an individual, which condition is attributable at least in part to the expression of a defective gene, said method comprising administering to said individual an effective number of autologous genetically altered cells, wherein said cells in their non-altered form are characterised by the subject defective gene, said genetic alteration comprising the introduction of at least one modification to said defective gene wherein said genetically altered cells are characterised by the capacity to produce an expression product or derivative, homologue, analogue, mutant or mimetic thereof which exhibits improved functional activity relative to the expression product produced by said defective gene.

Reference to a "defective gene" should be understood as a reference to any gene which is not fully functional in that it cannot perform the full range of functional activities which are performed by a non-defective form of the subject gene. By "functional activities" in this context is meant both the expression of a protein product and the performance of regulatory functions (which do not necessarily involve the production of an expression product). It should be understood that the gene, to the extent that it would normally exhibit more than one function may exhibit a defect only in relation to some of its functions. Where such defects occur, the gene is nevertheless to be understood as falling within the scope of "defective gene" as provided herein. The present invention is exemplified herein with respect to a defective dystrophin gene, the defect in respect of which results in the production of non-functional dystrophin thereby leading to the development of a dystrophic condition, in an affected individual, due to an effective dystrophin deficiency. It should be understood, however, that the exemplification provided herein is not intended as a limitation of the present invention which is directed to a method of treating any condition, attributable to the expression of a defective gene, via the genetic repair of autologous cells.

Accordingly, in one embodiment the present invention is directed to a method for treating dystrophin deficiency in an individual, which deficiency is attributable at least in part to defective dystrophin production, said method comprising administering to said individual an effective number of autologous genetically altered cells, which cells in their non-altered form are characterised by a defective dystrophin gene, said genetic alteration comprising the introduction of at least one modification to the dystrophin encoding gene wherein said genetically altered cells are characterised by the capacity to produce dystrophin or derivative, homologue, analogue, mutant or mimetic thereof, which dystrophin exhibits improved functional activity relative to the expression product produced by said defective dystrophin gene.

The present invention is predicated on genetically repairing autologous cells which exhibit a defective gene thereby leading to irregularities and/or defects in the expression or other form of functioning of the gene. Since the defect which is the subject of repair occurs in a gene, it will be evident in an individual's genomic DNA. In this regard, the defect will therefore generally be evident in the DNA of all an individual's cells. However, it should also be understood that the defect may be one which, despite existing at the genomic level, is evident in only some cell lineages. For example, it may be a defect which, although not evident in an immature stem cell, is caused to occur due to an inadvertent genetic event which may occur during, for example, the differentiation of the cell along a particular lineage and/or at a particular stage of development. An example of this would be defects which are evident in rearranged immunoglobulin genes, which genes are only found (in the rearranged form) in cells which have become committed to the B cell lineage. To the extent that the present invention is exemplified with respect to dystrophin deficiency, said invention is predicated on genetically repairing cells which exhibit a defective dystrophin gene such that production of functionally active dystrophin or derivative, homologue, analogue, mutant or mimetic thereof is facilitated. The defect which is the subject of repair in accordance with the invention described herein should be understood as being one which occurs in the gene encoding dystrophin and leads to the production of a non-fully functional expression product as hereinafter defined. Accordingly, the subject defect is evident at the dystrophin locus of the subject individuals genomic DNA.

Reference to "cell" herein should be understood as a reference to any cell which exhibits the subject defect. Preferably the subject cell is one in which the non-defective form of the subject gene would ordinarily function, either constitutively or in response to a stimulus. In another preferred embodiment, the subject cell is an immature form of a cell in which the non-defective from of the subject gene would ordinarily function or a multipotential cell, such as a stem cell, which exhibits the capacity to differentiate to a cell in which the non-defective form of the subject gene would ordinarily function. In accordance with these preferred embodiments, and in the context of the exemplified dystrophin model, examples of cells which are preferably the subject of repair include, but are not limited to, muscle cells (myocytes) or precursor muscle cells, such as myoblasts, or stem cells. However, it should be understood that the present invention is in no way limited to these preferred cells and may, therefore, utilise any suitable cell type.

In one preferred embodiment of the present invention, therefore, there is provided a method for treating dystrophin deficiency in an individual, which deficiency is attributable at least in part to defective dystrophin production, said method comprising administering to said individual an effective number of genetically altered autologous myoblasts, which myoblasts in their non-altered form are characterised by a defective dystrophin gene, said genetic alteration comprising the introduction of at least one modification to the dystrophin encoding gene wherein said genetically altered myoblasts are characterised by the capacity to produce dystrophin or derivative, homologue, analogue, mutant or mimetic thereof, which dystrophin exhibits improved functional activity relative to the expression product produced by said defective dystrophin gene.

In another preferred embodiment there is provided a method for treating dystrophin deficiency in an individual, which deficiency is attributable at least in part to defective dystrophin production, said method comprising administering to said individual an effective number of genetically altered autologous stem cells, which stem cells in their non-altered form are characterised by a defective dystrophin gene, said genetic alteration comprising the introduction of at least one modification to the dystrophin encoding gene wherein said genetically altered stem cells are characterised by the capacity to produce dystrophin or derivative, homologue, analogue, mutant or mimetic thereof, which dystrophin exhibits improved functional activity relative to the expression product produced by said defective dystrophin gene.

Reference to "administering" to an individual an effective number of genetically altered cells should be understood to include reference to either introducing into the individual an ex vivo population of cells which comprise repaired cells or else introducing into the individual materials required to repair a cell located in vivo, and preferably located in situ. With respect to this latter embodiment, the cell may be one which has always been present in the individual (that is, it has never been removed from the individual) or it may be one which was previously located ex vivo and has been introduced into the individual whereby in vivo repair of that cell will subsequently be effected. In this regard, it may be desirable to manipulate, culture, mark or otherwise treat the cell ex vivo in order to prepare it for genetic repair but to conduct the actual step of genetic repair in the in vivo, and even more preferably in situ, environment.

In accordance with the present invention, the subject cells are autologous cells which have been isolated, genetically repaired ex vivo and transplanted back into the subject individual. However, it should be understood that the present invention nevertheless extends to the use of cells derived from any other suitable source where the subject cells exhibit the same major histocompatability profile as the individual who is the subject of treatment. Accordingly, such cells are effectively autologous in that they would not result in the histocompatability problems which are normally associated with the transplanting of cells exhibiting a foreign MHC profile. Such cells should be understood as falling within the definition of "autologous". For example, under certain circumstances it may desirable, necessary or of practical significance that the subject cells are isolated from a genetically identical twin, or from an embryo generated using gametes derived from the subject individual. The cells may also have been engineered to exhibit the desired major histocompatability profile. The subject cells may have been freshly isolated from the individual prior to undergoing repair or they may have been sourced from a non-fresh source, such as from a culture (for example, where cell numbers were expanded and/or differentiation effected) or a frozen stock of cells, which had been isolated at some earlier time point either from the individual or from another source. It should also be understood that the subject cells, prior to undergoing gene repair, may have undergone some other form of treatment or manipulation, such as but not limited to, purification or modification of cell cycle status. Accordingly, the subject cell may be a primary or a secondary cell. A primary cell is one which has been isolated from an individual. A secondary cell is one which, following its isolation has undergone some form of in vitro manipulation, such as immortalisation or the prepartion of a cell line, as hereinbefore described.

That the genetically altered cells are characterised by the "capacity to produce" an expression product should be understood to mean that these cells, once repaired, can produce the subject expression product constitutively or could produce the subject expression product if appropriately stimulated. However, it should also be understood that the subject cell may be of an immature phenotype, such as a stem cell or, in accordance with the exemplified model, a myoblast, which may be required to undergo some degree of differentiation before it can produce the expression product either constitutively or in response to a stimulus. Such cells should nevertheless understood to fall within the scope of cells which are characterised by the "capacity to produce" the subject expression product.

In terms of the exemplified embodiment provided herein, reference to "dystrophin deficiency" should be understood as a reference to an individual exhibiting levels of dystrophin which are lower than that required to fully maintain dystrophin dependent functions and/or where the dystrophin expression product otherwise exhibits either amino acid sequence variation or other form of structural variation from wild type dystrophin. By "wild type dystrophin" is meant fully functional dystrophin. It should be understood that the subject dystrophin deficiency may be due either to a dystrophin gene defect which results in little or no dystrophin production or to a dystrophin gene defect which results in a structurally or functionally defective expression product. For example, dystrophin gene expression product levels in an individual may appear normal, however, if the subject expression product exhibits defects in the cysteine-rich and/or C-terminal regions then that expression product may exhibit structural weaknesses which would lead to muscle weakness in an affected individual. Accordingly, such an individual is nevertheless regarded as exhibiting dystrophin deficiency within the scope of the present invention. In another example, a given dystrophin defect may lead to unwanted symptoms in a localised region of the individual's body but not in another region of the body (for example, a given defect may lead to unwanted symptoms in muscle fibres but not in the brain, where dystrophin is also, found, or vice versa). Such an individual similarly falls within the scope of the present invention in that the individual is defined as suffering from dystrophin deficiency albeit a localised deficiency. In still yet another example, an individual may express a form of dystrophin which does not immediately lead to the development of symptoms characteristic of dystrophin deficiency, such as muscle weakness. However, the levels of dystrophin in such an individual nevertheless fall within the scope of the definition of dystrophin deficiency herein on the basis that the subject dystrophin deficiency is characterised by the production by that individual of an atypical form of dystrophin.

Reference to "dystrophin" should be understood as a reference to all forms of dystrophin or functional derivatives, homologues, analogues, mutants or mimetics thereof. Without limiting this definition in any way, dystrophin is the cytoskeletal protein which is found, inter alia, in muscle fibres, brain and other cells. The dystrophin protein comprises four distinct domains, being an N-terminal domain, a helical rod domain, a cysteine-rich domain and a C-terminal domain. Its functional role includes, but is not limited to, a structural role in maintenance of myofibre stability and integrity in myogenic cells and other cell types exhibiting a structural function. It is thought that the cysteine-rich and the C-terminal domains link with the extracellular matrix via β-dystroglycan and cyntrophin and also bind with dystrophin-associated glycoproteins. The N-terminal domain is known to associate with actin in myogenic cells. Reference to "dystrophin" should also be understood to encompass reference to derivatives of dystrophin including variants such as polymorphic or splice variants and dystrophin isoforms. In this regard, reference to "dystrophin" therefore includes reference to the shorter dystrophin isoforms which can be found in non-muscular tissue. For example, isoforms such as the 71 kDa dystrophin isoform found in glial cells and the 260 kDa form found in retinal epithelium are herewith encompassed. Without limiting the present invention in any way, these dystrophin isoforms generally comprise both the cysteine-rich and the C-terminal domains.

The dystrophin protein is encoded by the dystrophin gene (herein interchangeably termed "dys" or "dystrophin encoding gene"). Reference to dys should be understood to encompass reference to all nucleic acid molecule forms of dys, including for example all allelic, polymorphic and isoformic variants. In terms of the genomic dys sequence, it should also be understood to encompass any regulatory elements (such as promoters or enhancers) which regulate the expression of dys and includes any regulatory or protein encoding regions which exist at a location other than between the dys genomic DNA transcription initiation and termination sites. Further, to the extent that any region of dys may also contribute either structurally or functionally to the production of a non- dystrophin protein, these regions nevertheless fall within the scope of the present definition of dys. Dys contains 79 exons distributed throughout 2.5 million base pairs of chromosome Xp21.2. The introns occupy 99.4% of the gene sequence with the fully processed transcript being only 14kb. The critical cysteine-rich and cyntrophin binding domain towards the C-terminal of dystrophin are encoded on exons 62-72 and exon 74 of dys respectively. Expression of the gene is complex and occurs in a cell specific and developmentally regulated manner with at least seven independent promoters known throughout the gene. Reference to a "defective gene" should be understood as a reference to a gene which, when expressed, leads to the production of a non-fully functional expression product or non-fully functional regulatory function. By "non-fully functional" is meant that the expression product or regulatory function, if any, of the defective gene does not exhibit the same range or degree of functional activities, structural properties or regulatory functions that the expression product or regulatory function of the non-defective form of that gene would exhibit. For example, in terms of the embodiment exemplified herein, the dystrophin peptide from boys with Duchemie Muscular Dystrophy is a severely truncated form of the normal peptide, in which the peptide's cysteine-rich domain and/or C-terminal domain are missing or disrupted. These C-terminal truncations lead to severe degeneration of the muscle due to disruption of the binding regions of the mature dystrophin peptide thereby leading to instability of the myofibre plasma membrane. In another example, Becker's Muscular Dystrophy results from large truncations of the dystrophin protein, generally in the helical rod domain. The dystrophic condition developed here is less severe than that observed in Duchenne Muscular Dystrophy due to preservation of the dystrophin C- terminal. Nevertheless, both conditions provide examples of the production of an expression product which is not fully functional. Reference to a "defective gene" should also be understood to encompass a gene, the defect in which prevents the production of any form of expression product, per se.

The present invention is directed to facilitating the production of an expression product which exhibits improved functional activity, structural properties or regulatory functions relative to the expression product of the defective gene, by genetically altering the defective gene such that it is repaired. By "improved" is meant that the molecule produced by expression of the repaired gene exhibits:

(i) one or more functional activities or structural properties not exhibited by the expression product, if any, produced by the defective gene;

(ii) an increase in the degree of one or more existing functional activities or structural properties which are exhibited by the expression product of the defective gene. Accordingly, although it is preferable that the expression product derived from the repaired gene is fully functional in that it exhibits the same range and degree of functional activities and structural properties as the expression product which would be produced by a non- defective form of the gene, it should be understood that the production of a relative improvement, within the context detailed above, falls within the scope of the present invention.

Reference to "produce" should be understood as a reference to the expression (i.e. transcription and translation) a gene, being either a defective or a non-defective gene.

The term "derivatives" of the subject expression product includes fragments, variants (such as alleles), parts and portions. Derivatives include one or more insertions, deletions or substitutions of amino acids. Amino acid insertional derivatives include amino and/or carboxylic-terminal fusions as well as intrasequence insertions of single or multiple amino acids. Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site. Random insertion is also possible with suitable screening of the resulting product. Deletional variants are characterised by the removal of one or more amino acids from the sequence. Substitutional amino acid variants are those in which at least one residue of the sequence has been removed and a different residue inserted in its place. Additions to amino acid sequences include fusions with other peptides or polypeptides. This may be desirable, for example, to facilitate the co-expression of both the expression product and a molecule which supports the continued proliferation of the genetically repaired cells.

Derivatives also include fragments having particular biological or structural regions of dystrophin. A "mimetic" should be understood as a molecule which exhibits at least some of the biological or structural activity of the expression product.

Preferably the expression product is a human expression product, however, the invention also extends to homologues of the subject human expression product. A homologue as contemplated herein includes expression products exhibiting a sequence of a non-human species. For example, in one embodiment "homologues" of dystrophin include the alteration of a defective dys gene to produce a form of dystrophin which is normally found in a species other than the species which is the subject of treatment.

In terms of the particular form of expression product which is produced following genetic repair, this is likely to be determined when one considers the nature of the existing defects in the defective gene and the likely ease and efficiency with which one or more modifications can be introduced to render the gene capable of producing a functionally active expression product as hereinbefore defined.

Production of an expression product which exhibits improved functional activity relative to the expression product produced by the defective gene is achieved, in accordance with the method of the present invention, by genetically altering a defective gene such that an improved expression product, as hereinbefore defined, is produced. In this regard, reference to "genetic alteration" comprises the introduction of at least one modification to the nucleic acid molecule encoding the expression product. As hereinbefore defined, reference to "gene" includes reference to both protein encoding and regulatory regions of the subject gene. Accordingly, the subject modification may be introduced to one or both of these regions in the form of single or multiple modifications. Reference to "modification" is intended to encompass any form of nucleic acid modification such as, but not limited to:

(i) insertion or other form of introduction or association of a nucleic acid molecule with the subject gene such as a short fragment, individual nucleotides, amplicon, synthetic DNA peptide or an entire gene or portion of a gene.

(ii) deletion of one or more nucleotides or nucleic acid fragments from the subject gene.

(iii) mutation of one or more nucleotides of the subject gene. The present invention is exemplified with respect to the dystrophin gene defects which are known to lead to the development of Duchenne Muscular Dystrophy and Becker's Muscular Dystrophy. However, this is not intended as a limitation on the application of the present invention to the correction of any genetic defects other than those leading to Duchenne Muscular Dystrophy or Becker's Muscular Dystrophy. Similarly, exemplification of the present invention in the mdx mouse model, which is an accepted and standard model for the study of muscular dystrophy, is not intended as a limitation on the application of the method of the present invention to other species. In this regard, reference to "individual" herein should be understood to include reference to a mammal such as but not limited to human, primate, livestock animal (e.g. sheep, cow, horse, donkey, pig), companion animal (e.g. dog, cat), laboratory test animal (e.g. mouse, rabbit, rat, guinea pig, hamster), captive wild animal (e.g. fox, deer). Preferably the mammal is a human or primate. Most preferably the mammal is a human.

Without limiting the present invention to any one theory or mode of action, in Duchenne Muscular Dystrophy 96% of the cases involve frame shift or nonsense mutation of the dystrophin gene, with the remainder involving in frame deletions of the cysteine-rich and C-terminal domains. 65% of Duchenne Muscular Dystrophy cases involve a gene which exhibits gene rearrangements (predominantly frame shift, deletions or duplications) while the remaining 35 > have a dystrophin gene exhibiting either nonsense mutations or mutations that effect transcriptional splice sites. A small proportion of Duchenne Muscular Dystrophy individuals exhibit in frame dystrophin gene rearrangements which involves the two critical C-terminal binding domains. In the milder Becker's Muscular Dystrophy, 85% of cases are characterised by deletions with the remainder exhibiting a variety of point mutations. The Becker Muscular Dystrophy dystrophin gene mutations mostly preserve the C-terminal reading frames with most affecting the helical rod domain by in frame deletions or misense point mutations. However, the full range of genetic errors which may require repair is broad and includes point mutations, frame shift mutations, deletions, insertions or any other gene rearrangements. In a preferred embodiment, the dystrophin gene defect which is the subject of repair is a frame shift, a nonsense mutation or an in frame deletion of the cysteine-rich and/or C-terminal domains Accordingly, in one preferred embodiment there is provided a method for treating dystrophin deficiency in an individual, which deficiency is attributable at least in part to defective dystrophin production, said method comprising administering to said individual an effective number of genetically altered autologous cells, which cells in their non-altered form are characterised by a frame shift, nonsense mutation or in frame, deletion of the dystrophin gene region encoding the cysteine-rich and/or C-terminal domains of dystrophin, said genetic alteration comprising the introduction of at least one modification to said dystrophin encoding gene wherein said genetically altered cells are characterised by the capacity to produce dystrophin or derivative, homologue, analogue, mutant or mimetic thereof, which dystrophin exhibits improved functional activity relative to the expression product produced by said defective dystrophin gene.

Still more preferably there is provided a method for treating dystrophin deficiency in an individual, which deficiency is attributable at least in part to defective dystrophin production, said method comprising administering to said individual an effective number of genetically altered autologous cells, which cells in their non-altered form are characterised by a frame shift, nonsense mutation or in-frame deletion of the dystrophin gene region encoding the cysteine-rich and/or C-terminal domains of dystrophin, said genetically alteration comprising the introduction of at least one modification to one or more of exons 62-72 or 74 of said dystrophin encoding gene wherein said genetically altered cells are characterised by the capacity to produce dystrophin or derivative, homologue, analogue, mutant or mimetic thereof, which dystrophin exhibits improved functional activity relative to the expression product produced by said defective dystrophin gene.

In another preferred embodiment there is provided a method of treating dystrophin deficiency in an individual, which deficiency is attributable at least in part to defective dystrophin production, said method comprising administering to said individual an effective number of genetically altered autologous cells, which cells in their non-altered form are characterised by a nucleotide deletion or point mutation of the dystrophin gene region encoding the helical rod domain of dystrophin, said genetic alteration comprising the introduction of at least one modification to said dystrophin encoding gene wherein said genetically altered cells are characterised by the capacity to produce dystrophin or derivative, homologue, analogue, mutant or mimetic thereof, which dystrophin exhibits improved functional activity relative to the expression product produced by said defective dystrophin gene.

In accordance with these preferred embodiments it is still more preferable that the subject autologous cells are autologous stem cells or autologous myoblasts.

In an even more preferred embodiment, the subject repair takes the form of insertion of a nucleic acid molecule or derivative or analogue thereof, which molecule comprises at least one nucleotide. In a most preferred embodiment, where the subject defect is an unwanted deletion, the subject repair takes the form of insertion of said nucleic acid molecule or derivative or analogue thereof at the deletion interface. To the extent that the subject defect is a mutated nucleotide, the subject repair preferably takes the form of a point mutation which is achieved via the insertion of a nucleic acid molecule or derivative or analogue thereof to the genomic sequence.

The mechanism utilised to introduce the subject modification may be any suitable molecular technique including, but not limited to, homologous recombination or chimeroplasty. In accordance with a preferred aspect of the present invention, the subject repair takes the form of the insertion of a nucleic acid molecule or derivative or analogue thereof to the subject cells genome. Even more preferably, said insertion is achieved utilising the technique of small fragment homologous recombination.

Accordingly, there is provided a method for treating a condition in an individual, which condition is attributable at least in part to the expression of a defective gene, said method comprising administering to said individual an effective number of autologous genetically altered cells, which cells in their non-altered form are characterised by the subject defective gene, said genetic alteration comprising the introduction of a nucleic acid molecule or derivative or analogue thereof to the subject gene by small fragment homologous recombination, wherein said genetically altered cells are characterised by the capacity to produce an expression product or derivative, homologue, analogue mutant or mimetic thereof which exhibits improved functional activity relative to the expression product produced by said defective gene.

In a preferred embodiment there is provided a method for treating dystrophin deficiency in an individual, which deficiency is attributable at least in part to defective dystrophin production, said method comprising administering to said individual an effective number of genetically altered autologous cells, which cells in their non altered form are characterised by a defective dystrophin gene, said genetic alteration comprising the introduction of a nucleic acid molecule or derivative or analogue thereof to the dystrophin encoding gene by small fragment homologous recombination, wherein said genetically altered cells are characterised, by the capacity to produce dystrophin or derivative, homologue, analogue, mutant or mimetic thereof, which dystrophin exhibits improved functional activity relative to the expression product produced by said defective dystrophin gene.

In one preferred embodiment, said defect is a frame shift, a nonsense mutation or an in frame deletion of the dystrophin gene region encoding the cysteine-rich and/or C-terminal domains. Still more preferably, said nucleic acid molecule(s) are introduced to one or more of exons 62-72 or 74.

In another preferred embodiment, said defect is a nucleotide deletion of point mutation of the dystrophin gene region encoding the helical rod domain.

In accordance with these preferred embodiments it is still more preferably provided that the subject autologous cells are autologous stem cells or autologous myoblasts.

Introduction of the nucleic acid molecule or derivative or analogue thereof to the subject cell may also be achieved by any suitable means including, but not limited to, use of lipofection, cationic lipid, polyethyleneimine, calcium chloride, biolistic delivery, electric pulse mediated means (electroporation), micro-injection or other means of introducing DNA into the cystoplasmic, nuclear or mitochondrial compartments of the subject cell. The choice of mechanism will depend largely on the specific form of genomic repair which is required, the location of the cell to be repaired (i.e. in vitro, in vivo or in situ) and the functional and phenotypic characteristics of the subject cell.

The nucleic acid molecule or derivative or analogue thereof which is utilised to repair a defective gene may comprise one or more nucleotides and may be derived from natural or recombinant sources or may be chemically synthesised. Methods for producing these molecules would be well known to those skilled in the art. The subject nucleic acid molecule may be ligated or fused or otherwise associated with a nucleic acid molecule encoding another entity such as, for example, a signal peptide. It may also comprise additional nucleotide sequence information fused, linked or otherwise associated with it either at the 3' or 5' terminal portions or at both the 3' and 5' terminal portions. The nucleic acid molecule may also be part of a vector designed to facilitate its delivery to a target cell. In another example, it may be desirable that the subject nucleic acid molecule which is utilised with the homologous recombination technique comprises a marker in order to enable its detection and/or selection.

In this regard, reference to "derivatives" of the subject nucleic acid molecule should therefore be understood to include reference to fragments, parts, portions, chemical equivalents, mutants, homologues and mimetics from natural, synthetic or recombinant sources. Preferably, these derivatives are functional in that they exhibit any one or more of the functional activities of nucleotides or nucleic acid sequences. The derivatives of the subject nucleotides or nucleic acid sequences include fragments having particular parts of a nucleotide or nucleic acid sequence which is fused to other proteinaceous or non- proteinaceous molecules. Analogues contemplated herein include, but are not limited to, modifications to the nucleotide or nucleic acid sequence such as modifications to its chemical make-up or overall conformation. This includes, for example, modification to the manner in which nucleotides or nucleic acid sequences interact with other nucleotides or nucleic acid sequences such as at the level of backbone formation or complementary base pair hybridisation. The biotinyalation of a nucleotide or nucleic acid sequence is an example of a functional derivative which may be useful, for example, as a nucleic acid molecule which can be efficiently detected by virtue of the marker.

It should be understood that the method of the present invention can either be performed in isolation to treat the condition which is characterised by expression of the defective gene, for example dystrophin deficiency where the defective gene is the dys gene, or it can be performed together with one' or more additional techniques designed to facilitate or augment the subject treatment. For example, the gene repair aspect of the present invention may take the form of the application of two or more distinct repair mechanisms (for example, the method of the present invention envisages the application of small fragment homologous recombination together with anti-sense induced exon skippingor cytokine mediated methods to enhance uptake of repaired cells in target tissues. In another example, it would be envisaged that the method of the present invention could be applied together with the application technology designed to render a cell particularly receptive to the genetic alteration mechanism which is elected for use. In yet another example, it may be desirable to render the subject individual receptive to the receipt of genetically altered cells. For example, to the extent that the subject altered cells are stem cells it may be envisaged that the recipient individual would also undergo some form of cytokine therapy in order to support the proliferation and differentiation of the subject stem cells along a particular lineage, such as the myogenic lineage where the defective gene is a dystrophin gene.

In terms of the embodimemnt exemplified herein and still without limiting the present invention to any one theory or mode of action, repair of the dystrophin gene in cells cultured from a dystrophic individual by single fragment homologous recombination, followed by autologous cell transfer back to the individual's muscle results in the development of fibres which express functional dystrophin from an intrachromosomal locus under the control of endogenous genetic regulatory mechanisms. This invention can be applied to dystrophic patients with dystrophin gene deletions by the insertion of 1 to 2 bp at the deletion interface and, more directly, to point mutations by standard recognition. Successful application of single fragment homologous recombination-autologous recombination preferably results in the restoration of the entire dystrophin protein sequence. In individual's whose dystrophin gene mutation involved out of frame deletion, the insertion of a maximum 2 nucleotides restores the downstream reading frame and consequently the gene product's C-terminal regions. The autologous cell transplantation approach significantly deals with immunorejection issues and provides a rational solution to the treatment of dystrophin deficiency. It is expected that the method of the present invention could extend the lifespan of a boy with Duchenne Muscular Dystrophy by up to 20 years with a single treatment.

As detailed earlier in relation to the embodiment exemplified herein, reference to "dystrophin deficiency" in an individual is a reference to an individual exhibiting levels of dystrophin which are lower than that required to fully maintain dystrophin dependent functions and/or where an individual's dystrophin otherwise exhibits functional or structural variations relative to wild type dystrophin. Accordingly, the method of the present invention is particularly useful in relation to the therapeutic and/or prophylactic treatment of disease conditions characterised by dystrophin deficiency. A disease condition "characterised" by dystrophin deficiency should be understood as a condition at least one symptom of which is directly or indirectly due to the existence of a dystrophin deficiency as hereinbefore defined. In one particular aspect, the subject condition is muscular dystrophy and still more particularly Duchenne Muscular Dystrophy or Becker's Muscular Dystrophy. Similarly, to the extent that the present invention is directed to the repair of defective genes other than the dystrophin gene, there is facilitated the therapeutic and/or prophylactic treatment of conditions at least one symptom of which is directly or indirectly due to the existence of the defective gene as hereinbefore defined.

Accordingly, another aspect of the present invention is directed to the therapeutic and/or prophylactic treatment of a disease condition in an individual, which disease condition is characterised by the expression of a defective gene, said method comprising administering to said individual an effective number of genetically altered autologous cells, which cells in their non-altered form are characterised by the subject defective gene, said genetic alteration comprising the introduction of at least one modification to said defective gene wherein said genetically altered cells are characterised by the capacity to produce an expression product or derivative, homologue, analogue, mutant or mimetic thereof which exhibits improved functional activity relative to the expression product produced by said defective gene.

In a preferred embodiment the present invention is directed to the therapeutic and/or prophylactic treatment of a disease condition, which disease condition is characterised by a dystrophin deficiency attributable at least in part to defective dystrophin production, said method comprising administering to said individual an effective number of genetically altered autologous cells, which cells in their non-altered form are characterised by a defective dystrophin gene, said genetic alteration comprising the introduction of at least one modification to the dystrophin encoding gene wherein said genetically altered cells are characterised by the capacity to produce dystrophin or derivative, homologue, analogue, mutant or mimetic thereof, which dystrophin exhibits improved functional activity relative to the expression product produced by said defective dystrophin gene.

Preferably, said cells are stem cells or myoblasts.

Without limiting the present invention to any one theory or mode of action, it has been determined that the restoration of approximately 20% of wild type levels of normal dystrophin will correct muscle pathology in a dystrophic individual. • In terms of disease conditions attributable to other defective genes, it is within the skill of the person of skill in the art to determine, in a routine manner, the levels of expression of the genetically altered gene product which is required to attain the desired effect (for example, testing by physiological means the effect of the treatment).

In this regard, an "effective number" means that number necessary to at least partly attain the desired effect, or to delay the onset of, inhibit the progression of, or halt altogether, the onset or progression of the particular condition being treated. Such amounts will depend, of course, on the particular conditions being treated, the severity of the condition and individual patient parameters including age, physical conditions, size, weight and concurrent treatment. These factors are well known of those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is preferred generally that a maximum dose be used, that is, the highest safe dose according to sound medical judgement. It will be understood by those of ordinary skill in the art, however, that a lower dose or tolerable dose may be administered for medical reasons, psychological reasons or for virtually any other reasons.

It should be understood that although the method of the present invention is predicated on the introduction of genetically repaired cells to an individual suffering a condition as herein defined, it is not necessarily the case that every cell of the population introduced to the individual will have been repaired and/or will exhibit the capacity to produce the expression product. The present invention is achieved provided at least a portion of the cells thereby introduced constitute the "effective number" as defined above. Although in a particularly preferred embodiment a population of cells will undergo genetic alteration followed by the identification of successfully altered cells subsequent to their clonal expansion and reintroduction to the subject individual, it will not always necessarily be the case that such a procedure is performed. In some instances it may be more practical and efficient to subject a population of cells to genetic alteration and provided that this population, as a whole, are shown to produce the requisite levels of the expression product, this population as a whole can be introduced into the subject individual without the prior removal of non-altered or non-successfully altered cells. Accordingly, reference to "an effective number", in this regard, should be understood as a reference to the total number of cells required to be introduced such that the number of repaired cells is sufficient to produce levels of expression product which achieve the object of the invention, being the treatment of the subject condition.

As detailed hereinbefore, genetic alteration of the subject cells can be performed in vivo, in situ or in vitro. In the latter situation, the subject cell will then require introduction into the subject individual. Where the cells are altered in vitro, the subject cells are preferably ones which were isolated from the individual to be treated (i.e. autologous cells). However, the present invention nevertheless extends to the use of cells sourced elsewhere, such as syngeneic cells from an identical twin or cells from an embryo which exhibit the same major histocompatability profile as that of the individual in question. To the extent that the cells are altered in vitro, the cells may be subsequently introduced into an individual by any suitable method. For example, cell suspensions may be introduced by direct injection or inside a blood clot whereby the cells are immobilised in the clot thereby facilitating transplantation. Routes of administration include, but are not limited to, intravenously, intraperitonealy, subcutaneously, intracranialy, intradermaly, intramuscularly, intraocularly, intrathecaly, intracerebraly, intranasaly, by infusion, orally, rectally via i.v. drip or implant. In accordance with the embodiment exemplified herein, to the extent that the individual is being treated for muscular dystrophy, an intramuscular route is particularly preferred. The subject cells may also be introduced by surgical implantation. This may be necessary, for example, where the cells exist in the form of a tissue graft or where the cells must be encapsulated prior to transplanting. The site of transplant may be any suitable site, for example, intramuscularly or at a location proximal to the subject muscle. Without limiting the present invention to any one theory or mode of action, where the cells are administered as an encapsulated cell suspension, the cells will coalesce into a mass. It should be understood that the cells may continue to divide following transplantation. It should also be understood that to the extent that an immature cell source is utilised, such as a stem cell or a myogenic cell, the subject cells would likely continue to both proliferate and differentiate following transplantation.

In one preferred embodiment there is provided a method for the therapeutic and/or prophylactic treatment of Duchenne Muscular Dystrophy said method comprising administering to said individual an effective number of autologous genetically altered cells, which cells in their non-altered form are characterised by a defective dystrophin gene, said genetic alteration comprising the introduction of at least one modification to the dystrophin encoding gene wherein said genetically altered cells are characterised by the capacity to produce dystrophin or derivative, homologue, analogue, mutant or mimetic thereof, which dystrophin exhibits improved functional activity relative to the expression product produced by said defective dystrophin gene. Preferably, said genetic alteration is a modification to the dystrophin gene region encoding the cysteine-rich domain and/or the C-terminal domain and even more preferably to one or more of exons 62-72 and/or 74. Still more preferably, said genetic alteration is the introduction of a nucleic acid molecule or derivative or analogue thereof by single fragment homologous recombination.

In another preferred embodiment there is provided a method for the therapeutic and/or prophylactic treatment of Becker's . Muscular Dystrophy, said method comprising administering to said individual an effective number of autologous genetically altered cells, which cells in their non-altered form are characterised by a defective dystrophin gene, said genetic alteration comprising the introduction of at least one modification to the dystrophin encoding gene wherein said genetically altered cells are characterised by the capacity to produce dystrophin or derivative, homologue, analogue, mutant or mimetic thereof, which dystrophin exhibits improved functional activity relative to the expression product produced by said defective dystrophin gene.

In accordance with this preferred embodiment said genetic alteration is a modification to the dystrophin gene region encoding the helical rod domain and even more preferably comprises the introduction of a nucleic acid molecule or derivative or analogue thereof by single fragment homologous recombination.

In accordance with this preferred embodiments, the subject cells are preferably stem cells or myoblasts.

In accordance with the method of the present invention, other proteinaceous or non- proteinaceous molecules may be co-administered either with the introduction of the repaired cells or subsequently thereto, such as during gene expression by the introduced cells. By "co-administered" is meant simultaneous administration in the same formulation or in different formulations via the same of different routes or sequential administration via the same or different routes. By "sequential" administration is meant a time difference of from seconds, minutes, hours or days between the introduction of these cells and the administration of the proteinaceous or non-proteinaceous molecules or the onset of dystrophin production and the administration of the proteinaceous or non-proteinaceous molecule. For example, it may be necessary to co-administer cytokines in order to facilitate differentiation of an immature cell type such as a stem cell or a myogenic cell. Alternatively, it may be necessary to co-administer molecules in order to stimulate dystrophin production, or, where gene expression occurs constitutively, it may be necessary to inhibit the proliferation of these cells once expression product levels have been normalised in recipients. It should be understood that co-administration is in no way limited to these examples.

In a related aspect of the present invention, the subject undergoing treatment or prophylaxis may be any human or animal in need of therapeutic or prophylactic treatment. In this regard, reference herein to "treatment" and "prophylaxis" is to be considered in its broadest context. The term "treatment" does not necessarily imply that a mammal is treated until total recovery. Similarly, "prophylaxis" does not necessarily mean that the subject will not eventually contract a disease condition. Accordingly, treatment and prophylaxis include amelioration of the symptoms of a particular condition or preventing or otherwise reducing the risk of developing a particular condition. The term "prophylaxis" may be considered as reducing the severity of the onset of a particular condition. "Treatment" may also reduce the severity of an existing condition or the frequency of acute attacks.

In a preferred embodiment the subject of the treatment is a mammal and still more preferably a human. As detailed hereinbefore, although the present invention is exemplified utilising a murine model, this is not intended as a limitation on the application of the method of the present invention to other species, in particular, human.

In terms of the embodiment exemplified herein, although the method of the present invention is particularly suited to the treatment or prophylaxis of muscular dystrophy it is not to be understood as being limited to the treatment of this condition. Rather, the method for the present invention can be utilised to treat any condition characterised by dystrophin deficiency.

Further examples of the present invention are more fully described in the following non- limiting examples.

EXAMPLE 1 THE MDX MOUSE MODEL FOR DMD - STRATEGY FOR REPAIR OF A

NONSENSE TRANSITION

The dystrophin defect in human DMD is emulated in the C57BL/10J mdx mouse, which likewise demonstrates a lack of dystrophin staining by muscle immunohistochemistry. Several strains of mdx mouse have been characterised, all containing point mutations which result in downstream codon reading frame shift and consequent disruption of the C-terminal in the dystrophin protein (Im et al., 1996). The most commonly used strain of mdx mouse expresses a nonsense C to T transition in exon 23 of the dys gene (Sicinski et al., 1989) at position 3185 (3185T) of the dys mRNA sequence. Whilst the entire dys mRNA sequence for the mdx mouse is known, relatively small amounts of fragmented data are available for the remaining 94.6% of dys gene sequence. There is available however, access to 800 bp of sequence data inclusive of exon 23 and the 3185 position from the ENTREZ database. From available sequence, a strategy has been designed for Single Stranded Short Fragment Homologous Replacement (SFHR) of the mdx-3X85T mutation with the wt-3185C allele (Fig 1). This repair strategy is supported by high resolution allele-specific PCR (Fig 2; 1/10⁵ detection frequency) and quantitative (Fig 6) PCR-RFLP mutation detection which are both able to differentially detect specific repair at the chromosomal locus distinct from the introduced PCR fragment (Product C) by use of a primer site (B) not present on the PCR fragment (Fig 1&2).

EXAMPLE 2 CELL TRANSPLANTATION THERAPY AS A TREATMENT STRATEGY FOR

DMD

Cells from inbred C57BL/10J mdx donors may be repaired at the dys locus and isografts of the repaired cells performed using littermate recipients. The rationale of this approach is emphasised by enhanced persistence of isografts in which extended periods of biologically significant wt dystrophin expression levels were achieved by using "totally histocompatible" myoblasts from C57BL/10J +/+ donors in C57BL/10J mdx/mdx mouse recipients (Nilquin et al, 1995). The importance of genetically corrected cell autografts in human DMD is further highlighted by failure of human MTT trials due to graft rejection (Mendell et al., 1995). The recent observation of mdx muscle remodelling by systemic injection of wt bone marrow and cultured myoblasts gives rise to the possibility that human DMD may be treated by bone marrow transplantation (Gussoni et al., 1999). Unlike the mdx mouse model in which the inbred C57BL/10J background allows isogeneic cell grafts, human BM transplants are mostly allogeneic with isografts only possible between identical twins. Graft persistence under these conditions requires continual immune suppression to prevent cell graft rejection. The compromised respiratory status of boys with DMD makes long term immunosuppression unviable and lends further significance to an autograft approach involving gene repair in the patient's own cells. Systemic administration of wt C57BL/10J BM to C57BL/10J mdx mice has been investigated and it has been found that even 2 months post transplantation, peripheral blood was virtually entirely composed of cells with a wt dys locus, indicating that near total replacement of mdx BM is possible (Fig 4). Muscle remodelling can be stimulated by LIF and myotoxins such as notexin or bupivacaine. If such remodelling is induced in mdx muscle after transplantation with wt dys (ie repaired) cells, then existing mdx muscle can only be remodelled by cells containing wt dys locus. This is thought to be equally applicable for intramuscular (i.m.) administration as it is for intravenous (i.v.) injection of wt dys locus cells.

EXAMPLE 3 DMD DYSTROPHIN GENE REPAIR BY SMALL FRAGMENT HOMOLOGOUS

RECOMBINATION (SFHR)

None of the strategies currently being developed to treat Dystrophic cells in DMD involve direct repair of the genetic defect in the subject's genome. Using the strategy of Single Stranded Small Fragment Homologous Replacement (SFHR), it has been possible to achieve dystrophin levels as high as 5% in mdx myoblast cultures (Fig 6). In the first series of experiments (Fig 5&6), different lipofection reagents were applied and it was found that Lipofectin was able to more efficiently promote SFHR (after just 1 application) than Lipofectamine (Fig 5). Further, a double SFHR application was performed to cultures 1&2 (giving cultures 3&4), which likewise improved SFHR efficiency with Lipofectamine (Fig 5&6) to a level in excess of that observed with Lipofectin. This observation was confirmed in a second separate experiment (Fig 7), in which cultured myoblasts from one male mdx mouse (control culture A) was repaired once (culture la), twice (culture lb) and 3 times (lc), showing a significant improvement in SFHR efficiency after the first application (Lipofectamine/Plus reagent).

This work also showed that allele-specific PCR of mdx dys locus in the presence of the introduced PCR product (Fragment C), did not give rise to wt amplification product (Fig 6&7). With further optimisation, it is estimated that up to or greater than 10% recombination can be achieved with SFHR. Specifically, this can be achieved by variation of the Lipofection reagents, multiple SFHR applications or by electroporation. Incorporation of the E. coli derived recombination proteins such as RecA and RuvC in cell cultures has resulted in 10-fold increases in homologous recombination frequencies (Reiss et al., 1996). RecA protein is commercially available and may be incorporated into the SFHR protocol to further improve gene repair efficiency. The optimisation of SFHR efficiency provides a platform for clonal enrichment of repaired cells by serial dilution and expansion of transfected cultures so that as close to 100% of cells in such cultures as is possible will contain a repaired (wt) dys locus. This will provide the basic stock of repaired cells for systemic or intramuscular isografts in C57BL/10J littermates that will provide a strong basis for autogeneic transplantation of genetically corrected cells in human DMD.

As a final testament to the applicability of SFHR to gene repair in the mdx mouse, SFFfR was applied in vivo to mdx mice whose tibialis anterior muscles were treated with bupivacaine to promote remodelling. The RHS ta was subjected to SFHR, whilst the contralateral LHS ta was injected with transfection media without wt DNA. This preliminary experiment showed repair at the mdx dys locus of all ta muscles subjected to SFHR with no repair at the contralateral (saline) ta muscle in the same animal (Fig 8). These experiments lend favourably to the persistence of the introduced wt PCR fragment in cells for at least 10 days after SFHR, which may give rise to further in vivo gene repair after the introduction of repaired cells to mdx recipients. The application of a novel high resolution allele specific RT-PCR technique which has been developed will determine/confirm expression status of repaired dys loci in all our SFHR experiments.

EXAMPLE 4

SMALL FRAGMENT HOMOLOGOUS REPLACEMENT IN THE MDX MOUSE

DYS GENE (MYOBLASTS AND BM STEM CELLS)

(i) Generation of Wild-Type mouse dystrophin DNA Fragment for SFHR

The mdx mouse expresses a C-terminal dystrophin defect analogous to the human DMD condition via the presence of a single C to T nonsense transition at position 3185 affecting the exon 23 region of the dystrophin mRNA sequence (Sicinski et al.., 1989). This mutation causes the acquisition of a premature termination codon and consequently, the premature termination of protein translation downstream of the mutation. A 603 bp region inclusive of 301 nucleotide positions (np) either side of the mutation locus of the C57BL/10J +/+ (wild-type; wt) mouse dystrophin gene (dys) is amplified by PCR using a proofreading polymerase system (fragment/PCR Product C, Fig 1; dys-3X85wt) to minimise misincorporation error (Kapsa et al.,

2000). The amplified fragment is column purified and sequence checked by DNA sequencing to ensure the presence of the wild-type C at np3185.

(ii) Generation of mdx Bone Marrow Stem (BMS) Cells

Bone marrow is extracted from both femoris and tibialis of 120 male mdx C57BL/10J mice (8 weeks of age) by standard procedures and cultured in D- MEM/15% FCS media supplemented with Gin, pyruvate, uridine and LIF, conditions shown to facilitate BMS cell maintenance and expansion in vitro (Shih et /., 1999). (iii) Generation of mdx Myoblasts

Skeletal muscle is taken from the 120 male c57BL10J mdx mice (above) and myoblast cultures established. These cells are maintained in Hams/20% FCS medium according to standard procedures until sufficient numbers are generated for transfection. >

(iv) Generation of wt Bone Marrow Stem Cells (for Stages 2 & 3)

Bone marrow is extracted from both femoris and tibialis of 60 male wt C57BL/10J mice (8 weeks of age) and cultures set up as described for mdx mice.

(v) Generation of wt Myoblasts (for Stages 2 & 3)

Myoblasts are cultured from the 60 wt C57BL/10J mice as described above for mdx mice

(vi) Optimisation of SFHR (mdx Myoblasts and BM Stem Cells)

SFHR is performed according to established protocols (Kapsa et al., 2000), and optimisations performed likewise for BMS and myoblast cell cultures as follows:

Transfection Method:

Lipofectin Reagent: The optimal Lipofectin reagent for dys locus SFHR is determined by quantitative comparison of gene repair using AS-PCR or PCR RFLP (Figs 2 and 6). Commercially available reagents used include Lipofectin, Lipofectamine/Plus, Lipofectamine 2000, Fugene 6, Effectene, Superfect and Geneporter. Myoblasts transfected using Lipofectin reagents in serial transfections often show some signs of stress, particularly accelerated senescence. In other similar DNA transfer experiments, such stress is not evident for cells transfected by electroporation, by which membrane disruption can be minimised. Square-wave electroporation is evaluated as a possible vehicle for improvement of current SFHR efficiency levels.

Serial Multiple Transfection: In having performed double and triple serial cycles of SFHR (Fig 5 & 7), on mdx myoblasts, improved gene efficiency was visible on agarose gels subsequent to the initial SFHR. Densitometric quantification of two such cultures (cultures 3 & 4, Fig 5 & 6) revealed that approximately 5% of these cultures were composed of cells with a wt (repaired) dys locus. Employing the three most optimal transfection methods determined in the preceding section (above), serial SFHR is performed on mdx cells and quantified as shown in Figs 5 & 6.

E. coli Recombination Protein: The recombination protein RecA is incorporated during SFHR optimised as per the preceding 2 stages, and titrated to further optimise the SFHR process.

Quantitative Screening of Transfected Cultures

All transfected cultures are evaluated for wt dys locus and transcript content by the AS-PCR and PCR-RFLP methods outlined thus far and shown in Figs 3 to 8.

Expansion/Enrichment of Repaired Cultures

Limited Culture Transfection: A wt PCR Product C (transfection fragment) to cell ratio of 10⁶ to 1 results in a repair frequency of approximately 1%. Small diluted cultures containing 10 cells are transfected at this ratio. If any of these cultures contain repaired cells, then they will constitute a minimum 10% of the total cell population. Serial Dilution/Semi-Clonal Expansion: Transfected cultures are diluted to contain 1 to 5 cells per well. These are cultured to confluence and screened for wt dys locus, which will be quantified. These cultures are then diluted again to further enrich for cells containing wt dys locus until a minimum of 80% wt dys loci are present.

NOTE: On final enrichment of cultures, a passage is screened by Southern-RFLP for the presence of unincorporated PCR Product C (wt) and for non-homologous genomic insertion. Only cultures with no evidence of random insertion are used in subsequent stages. Some passages are allowed to form myotubes by growth in myoD for assessment of wt dystrophin expression.

EXAMPLE 5 MDX MOUSE MUSCLE REMODELLING BY INTRAMSCULAR (IM) INJECTION

The possibility that mdx cells containing dys locus repaired by SFHR (ie repaired cells) can remodel muscle is first assessed using cultured muscle precursors (myoblasts) and bone marrow stem (BMS) cells administered by i.m. injection. Both cell types are extracted from mdx mice 8 weeks of age and cultured. Alginate rods containing LIF (Austin et al. , 1997), constructed to release 5-10 ng of LIF per day are implanted adjacent to intended injection sites on the left and right ta muscles of the 60 mdx mice per cell type according to methods developed in our laboratory (Bower et al, 1997, Austin et al, 1997). The mdx mice are injected in their left ta with control cells derived from mdx littermates and in their right ta with the repaired mdx or wt cells. Five injections are made 1mm apart per muscle, each containing 4 x 10⁵ cells in the presence of 30 U/ml LIF in 10 μl. This gives a total of ~2 x 10⁶ cells per muscle injected, a number which facilitates dystrophin expression to therapeutic levels (Chamberlain, 1997). A further group of 30 mdx mice (per cell type) are injected with vehicle in the right ta and mdx C57BL/10 cells in their left ta (i.e. controls) and transplants involving repaired cells are compared to transplants involving wt cells. Mice (n=5) will be harvested at 2, 4, 8, 12, 20 and 36 weeks, ta muscles extracted, electrophysiology performed and muscles frozen on melting isopentante for cryostorage until required.

(i) Injection of Bone Marrow Stem cells (isografts): (Total of 90 mice)

Wild Type (wt) Bone Marrow Stem cells Repaired Bone Marrow Stem Cells (wt)

(ii) Injection of Myoblasts (isografts): (Total of 90 mice)

Wild Type (wt) myoblasts Repaired myoblasts (wt)

(iii) Detection of wt Gene in mdx Muscle

DNA is extracted from a portion of ta muscles removed from the transplanted mice (after muscle electrophysiology and histological determination of wt dystrophin expression), screened and quantified as described in preceding sections for the presence of wt dys locus. Southern RFLP is used to detect the presence and extent of unincorporated wt SFHR PCR Product C and random insertion.

(iv) Assessment of Dystrophin Expression in mdx Muscle

Repaired dystrophin expression is assessed histologically using anti-dystrophin antibodies and in situ Allele-Specific RT PCR. Following histology, RNA extracts are performed using some of the ta dissected from the transplanted mice. The RNA is screened and wt transcript quantified using allele-specific RT PCR and PCR- RFLP essentially as described for gene repair assays, but using oligo primers directed at exons other than 23 (not introns 22 and 23), up and downstream of the mdx mutation. mRNA in vitro (Quantitative):

Allele-specific PCR (semi quantitative) and PCR RFLP (quantitative) is performed using radiolabelled P³² dCTP and dys cDNA generated by gene specific RT-PCR. AS-RT-PCR products are quantified using ImageQuant software and normalised against GAPDH transcript. PCR-RFLP products are quantified using ImageQuant software as described above and shown in Figs 3 and 6.

Histology I (mRNA in situ):

Expression of wt or repaired dystrophin is assessed by allele-specific in situ RT PCR in the presence of DIG dUTP and visualised in situ by FITC labelled anti-DIG antibody.

Histology II (anti-Dvs Ab):

Dystrophin expression is assayed by anti-dystrophm antibody as routinely performed in our laboratory. Colour development is either by FITC or HRP- labelled secondary (anti-sheep) Ab. H&E staining enables assessment of dystrophic morphology. Large centrally nucleated fibres (dystrophic), peripherally nucleated fibres (normal) and small centrally nucleated fibres ("regenerating") are correlated with wt dystrophin expression and morphometric parameters compared using ImagePro software.

(iv) Measurement of Strength in Repaired mdx Muscle

All ta muscles from the grafted mdx mice undergo strength testing according to established procedures (Lynch et al., 1998). The muscles are immediately frozen for histological and nucleic acid analyses. EXAMPLE 7 MDX MOUSE MUSCLE REMODELLING BY SYSTEMIN (IN) INJECTION

The remodelling of mdx muscle using systemic (i.v.) administration of cultured myoblasts and BMS mdx cells containing SFHR-repaired dys locus is investigated. Both cell types are extracted from mdx mice 8 weeks of age, cultured and repaired as described earlier. After lethal and sub-lethal ablation of bone marrow by radiation therapy, recipient mdx mice are systemically injected with 2.5x10⁷ cells (suspended in 200 μl RPMI/2%FCS media) in the presence of 30 U/ml LIF into the tail vein. It has been determined that no BM ablation in hosts leads to minimal levels of BM replacement (data not shown). The right ta of each mouse with systemic injection is left as is, and the left ta of each mouse is injected with Bupivacaine 2 weeks after transplantation to induce tissue remodelling. A further group (n=30) per cell type have LIF rods implanted in the left ta prior to injection. As with the i.m. injected mice, these BM grafts are allowed to persist for 2, 4, 8, 12, 20 and 36 weeks after which heart, ta, diaphragm, liver and brain are harvested (5 mice per time point), electrophysiology performed (where appropriate) and tissues frozen on melting isopentane for cryostorage until required to assess efficiency and distribution of cell grafts. A further group of 30 mdx mice per cell type are injected with mdx cells (controls) and transplants involving repaired cells are compared to transplants performed with wt cells. Blood from transplant recipients are sampled (eye bleeds) monthly, the DNA extracted and tested for the presence of repaired/wt dys locus content as performed for and shown in Fig 4.

(i) Irradiation of mdx mice (400 and 1200 rads)

(ii) Injection of Bone Marrow Stem cells (isografts) (Total of 90 mice)

Wild Type (wt) Bone Marrow Stem cells Repaired Bone Marrow Stem cells (wt) (iii) Injection of Myoblasts (isografts) (Total of 90 mice)

Wild Type (wt) myoblasts Repaired myoblasts (wt)

(iv) Detection of wt Gene in mdx Tissues

DNA is extracted from a portion of the tissues removed from the transplanted mice (after muscle electrophysiology and histological determination of wt dystrophin expression), screened and quantified as described earlier.

(v) Assessment of Dystrophin Expression in mdx Tissues

Repaired dystrophin expression is assessed in the tissues extracted from the transplant recipient mdx mice as described earlier.

mRNA in vitro (Quantitative): See earlier

Histology I (mRNA in situ):

See earlier.

Histology II (anti-Dy^^b):

Dystrophin expression in all tissues is assayed by anti-dystrophin antibody. Colour development is either by FITC or HRP-labelled secondary (anti-sheep) Ab. H&E staining is performed on heart, diaphragm and ta for assessment of dystrophic morphology. Large centrally nucleated fibres (dystrophic), peripherally nucleated fibres (normal) and small centrally nucleated fibres ("regenerating") are correlated with wt dystrophin expression and morphometric parameters determined and compared using of ImagePro software. (vi) Measurement of Strength in Repaired mdx Muscle

Diaphragm, skeletal muscle and ta are subjected to electrophysiology as described earlier.

EXAMPLE 8

IN VIVO AND IN VITRO CORRECTION OF THE mdx DYSTROPHIN GENE

NONSENSE MUTATION BY SHORT FRAGMENT HOMOLOGOUS

REPLACEMENT MATERIALS & METHODS

(i) Mice for SFHR in Cell Culture

Two male C57BL/10J mdx mice, one 12 weeks of age, and the other a 9 day old male were obtained (Monash University Animal Services, Melbourne, Australia), exposed to a 12 hour day/night cycle and fed ad libitum until they were required for muscle culture. A further female C57BL/10 ScSn/ScSnY (wt) mouse 4 mths of age was obtained for cell culture and generation of wt DNA for gene repair. All animal handling was performed according to St Vincent's Hospital Animal Ethics Committee, protocol 26/99 in accordance with the Australian Code of Practice for the Care of Animals for Scientific Purposes (NHMRC).

(ii) Mice for SFHR in vivo

All procedures were approved by the Animal Experimentation Ethics Committee of the University of Melbourne and conformed to the guidelines for the care and use of experimental animals as described by the National Health and Medical Research Council of Australia. Throughout the experimental stages, animals were closely monitored to ensure there was no adverse reaction to the interventions. Four 4 month old male mdx mice (30-40g) were anesthetized with methohexitone sodium

(Brietal, Eli Lilly, USA. 40-60 mg/kg bodyweight i.p.) such that they were imresponsive to tactile stimuli. The Tibialis Anterior (TA) muscles of the left and right hindlimbs were surgically exposed and maximally injected with a total volume of -250 μl of 0.5% bupivacaine hydrochloride (Marcain, Astra, North Ryde, NSW, Australia) by one injection in each of the proximal, midbelly, and distal regions of the muscle. This was equivalent to, or exceeded, the maximum volume of bupivacaine that each TA muscle could hold, and caused degeneration of the entire muscle mass (unpublished observations). Intramuscular injection of bupivacaine to a muscle's holding capacity causes complete degeneration of muscle fibers within the first 2 days, followed by complete regeneration (Rosenblatt 1992). Following injection, the small skin incision was closed with

Michel clips (Aesculap, Germany) and swabbed with povidone iodine solution. At the completion of surgery, mice were returned to their cages where they were fed ad libitum and routinely monitored for condition.

(iii) Myoblast Cell Cultures

After asphyxiation with CO₂ and cervical dislocation, skeletal muscle was removed from the mdx mice and primary myoblast cultures prepared essentially as described elsewhere (Austin et al., 1992). The muscle tissue was finely minced using scissors in a small glass petri dish. The myoblast cultures were preplated twice for 1 hour in culture media containing Hams F10 (ICN Biomedicals) supplemented with 20% fetal calf serum (FCS), 2.5 ng/ml bFGF, 2 mM Gin, 100 units/ml of penicillin and 100 μg/ml streptomycin. All cultures were then grown in full growth media (T25 flasks) at 37°C and 5% CO₂ with daily half media changes until passage at >80% confluence (10⁶ cells).

Once cultures reached 80%) confluence the cells were detached from the flask using 0.05%) (w/v) trypsin in HBSS (which was neutralized by reintroduction to FCS immediately following cell detachment) and counted using a hemocytometer. The cells were divided into two aliquots each containing 5 x 10⁵ cells, pelleted at 400xg for 10 minutes and media aspirated and discarded. One of these pellets was resuspended in full growth media and grown for subsequent transfection and myotube differentiation, with the other frozen for DNA extraction. The cultures thus intended for transfection were grown to >80% confluence and split for transfection and differentiation.

(iv) Preparation of DNA for SFHR, mdx Control and assay

1. SFHR DNA.

DNA was extracted and column-purified from liver of the female wt mouse using the QIAamp Tissue DNA extraction protocol according to specification (Qiagen), suspended in 15 mM Tris.Cl pH 9.0 and stored at -20°C for DNA analysis. A 603 bp amplicon (Amplicon C) was generated by PCR using oligonucleotide primers C and D (Fig. 10, Table 1). The PCR reactions (100 μl) consisted of 50 ng of total DNA, 0.4 μM of each primer, 200 μM dNTPs, 1.5 mM MgCl₂, 0.5 units of Bio-X-

Act 3 '-5' proof-reading DNA polymerase system (Bioline/Fisher-Biotec) in buffer supplied by the manufacturer. The reactions were subjected to 29 cycles of 92°C/45s (denaturation), 65°C/2.5 min (annealing/extension) with an initial cycle of 92°C/2 min and 65°C/2.5 min (total 30 cycles) using a QuarterBath thermal cycler (Bartelt Instruments, Australia). Amplicon C PCR product was resolved from reagent components by electrophoresis (1% agarose, TAE), followed by ion exchange chromatography according to manufacturer's specification (QIAEX II system, Qiagen), and suspended in 15 mM Tris.Cl pH 8.8 at a final concentration of 0.1 μg/ul (0.27 pmol/μl).

2. DNA for mdx Control and assay of dys locus repair.

DNA for establishment of control mdx template was extracted from livers of the two male mdx mice used for establishing mdx cell cultures as described above for wt DNA. After gene repair by SFHR, cultures (10 cells) were harvested and half- passages used for extraction of DNA (QIAamp), which was then assessed for the presence of wt dys loci.

(v) Detection of wt (Repaired) dys Loci by Allele-Specific PCR (as-PCR)

AUele-specific PCR was performed using antisense oligonucleotide primer 'Dys wt AS-01' (3' mismatch for the mdx nucleotide) and sense primer B, (Table 1, Fig. 11 A). Using this protocol, a 415 bp analytical PCR product was only amplified from wt DNA (Fig 2A). Location of Primer B in an intronic region upstream of exon 23 and the mdx mutation locus, and in a gene region not present on Amplicon

C thus confers chromosomal locus specificity to the as-PCR assay used here. PCR was performed in 25 μl reactions as described above for wt DNA with the exception of a sequential increase in annealing temperature such that mdx DNA was not amplified (Fig. 11). Resolution of wt and mdx templates occurred by application of a 62.5°C annealing temperature.

The Tibialis Anterior (TA) and Vastus Lateralis (VL) muscles of the 9 day old mdx mouse were removed and frozen on melting N₂₍i₎-cooled isopentane for dystrophin immunohistochemistry, skeletal muscle DNA extraction and RNA extraction. DNA was extracted using the QIAamp Tissue DNA extraction protocol according to manufacturer's instruction (Qiagen), suspended in 15 mM Tris.Cl pH 9.0 and stored at -20°C for DNA analysis by allele-specific PCR and PCR-RFLP. This DNA (50 ng) and that extracted from transfected and untransfected cultures was subjected to allele specific PCR (as-PCR) as shown in Figure 11.

(vi) Detection of Repaired dys Loci by Quantitative PCR RFLP

PCR-RFLP based on an earlier assay (Asselin et al., 1994; Shrager et al., 1992), was performed on repaired cultures using a 3 '-modified oligonucleotide primer with allele-specific Mae III restriction enzyme site acquisition followed by PCR product restriction digestion (Fig. 11B). DNA was extracted from repaired mdx, native (umepaired) mdx and wt myoblast cultures, and PCR performed as described above (for Amplicon C) using primers A and D (Fig 10, Table 1). Reactions consisted of 50 ng of total DNA, 0.4 μM of each primer, 200 μM dNTPs, 1.5 mM MgCl , 2.5 units of Bio-X-Act 3 '-5' proof-reading DNA polymerase system (Bioline/Fisher-Biotec) in buffer supplied by the manufacturer. The reactions were performed as described for Amplicon C to generate a PCR product of 810 bp (Amplicon A) exclusive of the SFHR-introduced Amplicon C. The PCR product was resolved from reaction components by 1%> agarose gel electrophoresis and ion exchange column according to manufacturer's specification (Qiagen) and suspended in 15 mM TrisCl pH 8.8, at a concentration of 0.1 μg/μl. A secondary

PCR reaction was performed using 100 ng of Amplicon A, 0.4 μM of each primer (Dys Mae S-01 and Ex23 AS-01 Table 1, Fig 2B) in the presence of α³²P dCTP, otherwise as described above for Amplicon A. Digestion of this 104 bp amplicon (Dys Mae- 104) with Mae III with the mdx nucleotide at the dys_ locus, produces 60 (Band S, Fig 1 IB) and 44 (Band B, Fig 1 IB) bp bands. The 3' modification on the

Dys Mae S-01 primer results in site acquisition of a further Mae III site only in the presence of the wt nucleotide at the dys locus. Mae III digests the 44 bp Mae III digestion product of wt Dys Mae-X04 PCR product (Band B) into 2 fragments of 20 and 24 bp respectively (Band C, Fig 11B). Digests were performed at 55°C for 12 hrs, using 1 to 2 μg of Dys Mae-X04 product generated from repaired mdx, native mdx and wt myoblasts, 10 units of Mae III in buffered conditions as specified by the manufacturer (Promega). The digestion products were resolved on 16% non- denaturing acrylamide gels and visualised by phosphorimager (Molecular Dynamics). Quantification of wt loci was performed using ImageQuant software (Molecular Dynamics) and the relative volume densities of bands B (Basis, 44 bp) and C (Cut, 24/20 bp) as shown in Figure 2B. Incomplete digestion with the Mae III enzyme was quantified by comparison of volume densities of bands S (internal digestion Standard, 60 bp) and B in 100% wt template controls (Fig 11B). The possibility that the PCR-RFLP method could generate artefactual positive results owing to carry-over of Amplicon C in the DNA extracted from the repaired cells was also investigated (Lane mdx/C, Fig 11B). This control was achieved by addition of 10 ng of Amplicon C per 1 μl of mdx DNA template extracted from the control mdx cultures prior to Amplicon A amplification by PCR. This ratio of Amplicon C and mdx DNA represents the ratio expected if the entire 1 μg of Amplicon C that was used to transfect the mdx cultures was co-amplified with the repaired cells' genomic DNA (100 μl total volume).

(vii) Gene Repair at the dys Locus in mdx Cultures by SFHR

An Amplicon C/Lipofectamine/Plus Reagent complex was formed as follows: Heat-denatured (70°C/3 min) Amplicon C (1 μg, 2.7 pmol, 1.6 x 10¹² mol) was added to 100 μl of serum free OptiMEM (Gibco BRL) and allowed to equilibrate to room temperature for 10 minutes. To this was added 9 μl of Plus Reagent (Life Technologies) and after thorough mixing, the complex was allowed to equilibrate at RT for 30 mins. To a further 100 μl of OptiMEM was added 6 μl of Lipofectamine reagent (Life Technologies) and the mixture equilibrated at RT for

30 mins. The two mixtures were then combined and allowed to complex for a further 90 mins prior to transfection.

The Lipofectamine/Plus/ Amplicon C (6μl/9μl/lμg) complex was introduced to 5 x 10⁵ mdx myoblasts (1 cell to 3 x 10⁶ molecules of amplicon C) suspended in 1 ml of OptiMEM (Gibco BRL). The total volume was made up to 3.0 ml with

OptiMEM, cells transferred and allowed to settle in a TC25 flask (Nunc). The transfected myoblasts were propagated at 37°C in 5% C0 /air for a further 8 hrs after which the media were supplemented with 2.5 ml of fully supplemented HAMS-F10 growth media without antibiotic or fungicide, containing 20%) FCS, 2.5 ng/ml bFGF and 2 mM Gin. These transfected myoblast cultures were grown to

>80% confluence in full media, and split for DNA analysis (Culture la, Figure 12) and further transfection/differentiation. This procedure was serially repeated until mdx myoblast cultures which had been transfected 0, 1, 2, and 3 times had been generated, sampled for DNA (Cultures A, la, lb and lc respectively, Figure 12) and allowed to differentiate to myotubes for assessment of dystrophin expression. The possibility of artefactual positive results owing to carry-over of Amplicon C in the DNA extracted from the repaired cells was also investigated (Lane Al, Fig 12). This control was achieved by addition of 10 ng of Amplicon C per 1 μl of the DNA (100 μl) template extracted from repaired culture A (mdx) prior to asPCR analysis. This is the maximum ratio of Amplicon C and mdx DNA possible in the repaired cultures (la, lb and lc). This would only arise if the entire 1 μg of Amplicon C used to transfect Culture A was co-amplified with the repaired cultures' (la lb, and lc) genomic DNA (100 μl total volume).

Separate experiments were performed using Lipofectin and Lipofectamine (no Plus reagent, Fig 13) to assess relative efficiency of SFHR with different lipofection reagents.

Myotube differentiation was induced by growth of myoblasts in HAMS F10 media supplemented with 2 mM Gin, 100 units/ml of penicillin, 100 μg/ml streptomycin and 1% horse serum (HS) for two days, followed by supplemented HAMS F10 media with 2%> HS for a further 7 days. By the end of this incubation, more than 50% of the myoblasts appeared to have differentiated. Growth in 2%> HS beyond this point resulted in significant morbidity of cells regardless of whether they had been transfected or not (data not shown).

(viii) Assessment of dys Locus Expression by RT PCR

After differentiation of mdx myoblast cultures, mRNA was extracted from 10⁵ cells using the SV RNA extraction protocol (Promega). Total RNA was also extracted from a sample of mdx mouse VL, which had been ground on dry ice prior to extraction. First strand gene-specific reverse transcription was performed on 250 ng of mRNA using dys gene-specific oligonucleotide primer c3603-AS (dystrophin, see Table 1), and oligo dT (for GAP-DH), Superscript MMLV reverse transcriptase (Life Technologies) in buffered conditions specified by the manufacturer. An 803 bp dys transcript product was amplified from this gene specific cDNA using c3603- AS and c2801-S primers. Reactions (100 μl) consisting of 0.1 μM concentrations of each primer, 0.2 mM of each dNTP, 2 mM MgCl₂ and 5 units of Taq polymerase (Promega) were subject to 29 cycles of 93°C/1 min (denaturation), 57°C/45 sec (annealing) and 65.5°C/2 min (extension). A primary 3 min denaturation was performed during the first cycle to ensure optimal first stage denaturation. The details for GAP-DH RT PCR are described elsewhere (Reardon et al, 2000).

(ix) Gene Repair at the dys Locus in mdx Tibialis Anterior by SFHR

At 4 days post-bupivacaine injection, the mice were anaesthetized as described previously. Skin incisions were re-opened to facilitate the intramuscular injection of a SFHR cocktail consisting of 25 μg of Amplicon C complexed with Lipofectin at a ratio of 1:1 (m:v) in 0.9% NaCl, (final volume of 200 μl) into the right TA. An equivalent injection with DNA substituted with saline/Lipofectin vehicle was administered to the left TA muscle to provide a contralateral, no-DNA control.

Following injection, the skin incisions were re-sealed and re-swabbed, and the mice returned to their cages upon gaining full consciousness. The mice were harvested at 3 weeks following injection and both TAs removed and frozen on N₂₍i₎-cooled melting isopentane for immunohistochemical and gene repair analysis.

(x) Immunohistochemical Detection of Dystrophin Expression in Myoblasts and Muscle

Dystrophin expression in muscle and cultured myotubes was assessed using a polyclonal antibody raised in sheep against a 60 kDa dystrophin fusion protein as described elsewhere (Bower et al., 1997). Dystrophin on the slides was visualised by secondary HRP-conjugated rabbit anti-sheep antibody (DAKO) and colour developed with 3,3 Diaminobenzidine (DAB) using Sigma Fast DAB tablets (Sigma Chemicals) according to manufacturer's protocol. Endogenous peroxidase activity was blocked with 0.6% H₂0₂ in PBS prior to incubation with the primary antibody. Myotubes were grown in Labtekll 4-Chamber slides (Nunc) as described above, fixed for 15 minutes in 0.05% gluteraldehyde/PBS prior to immunohistochemical staining for dystrophin expression.

EXAMPLE 9

IN VIVO AND J3V VITRO CORRECTION OF THE mdx DYSTROPHIN GENE

NONSENSE MUTATION BY SHORT FRAGMENT HOMOLOGOUS

REPLACEMENT SFHR Gene Repair in mdx Mouse Myoblasts

The SFHR strategy used to repair the exon 23 C to T nonsense transition in mdx mouse myoblasts focused on an 876 bp region of the dystrophin locus (dys). Representing the entire available sequence data for this region of the dys gene, the sequence data encompassed partial nucleotide sequence from introns 22 and 23, and the entire exon 23 sequence (Fig. 10).

The location of primers A and B outside of Amplicon C (generated by primers C and D) provided access to the chromosomal locus in repaired cells exclusively of the introduced amplicon C (Figs. 10 and 11 A). This forms the basis for a novel allele-specific PCR (as- PCR) strategy that is able to resolve one wt dys locus from 10⁵ mdx loci (data not shown). The as-PCR strategy utilizes primer B with primer Dys wt AS-01 that has a 3 '-mismatch with the mutant T nucleotide at the mdx locus and results in differential amplification of wt template in a mixture of mdx and wt DNA (Fig. 11 A). This high-resolution technique was designed specifically for detection of the low level (-1%) gene repair expected from SFHR (Goncz et al., 1998) in the 10⁵ cells grown. Because of the exponential amplification of template using this method, the as-PCR strategy could only be considered semi quantitative. PCR-RFLP using allele-specific Mae III digestion was able to provide quantitative analysis of dys loci in repaired cell cultures (Fig. 11B). In contrast to the exponential nature of allele-specific PCR, PCR-RFLP has a linear range of wt loci detection in repaired (heterogeneous) cultures and can therefore be used as a quantitative measure. Prior to myoblast culture, as-PCR analysis of DNA extracted from the Tibialis Anterior (TA) of a 9 day old male mdx mouse showed a total absence of wt C at the mdx locus (Fig 11 A, mdx lane). Likewise, cells cultured from the remainder of the same mdx mouse's muscle were exclusively of the mdx genotype (Fig. 12, lanes A and Al). After a single transfection using Lipofectamine/Plus reagent of 3 such cultures established from the mdx mouse, as-PCR was able to detect successful replacement of mutant T nucleotide with the wt C at the mdx locus in 0.1%) to 5xl0^"4%> of cells (Fig. 12, cultures la, 2 and 3) which persisted for at least 28 days post repair (Fig. 12, culture 4). After two further serial applications of SFHR (cultures lb and lc, Fig. 12), improved repair efficiency was evident on agarose gels compared to just a single application of SFHR. This result was confirmed in further independent experiments involving myoblasts cultured from a single 4 mth old male mdx mouse (Fig. 13). In the latter experiment, SFHR efficiency in cultures transfected once with Lipofectamine (1 and la) was improved by a subsequent transfection (cultures 2 and 2a, Fig 13). Furthermore, SFHR efficiency with Lipofectin (Fig. 13, cultures 3 and 3 a) and Lipofectamine/Plus was better after single applications than with Lipofectamine alone. This result indicates that SFHR efficiency may be improved by variation of the transfection method and by multiple applications. In the second experiment (Fig. 13) the multiply transfected cultures (2 and 2a) were assayed for wt loci by PCR- RFLP, and showed a repair frequency of 15%) to .20%) using Imagequant software (Molecular Dynamics). Of the remaining cultures transfected in the second experiment (Fig 13), cultures 3 and 3a displayed the presence of wt loci in 1% to 2% of loci using the PCR-RFLP quantitative technique. Cultures 1 and la contained wt loci beyond the resolution limit of the PCR-RFLP strategy, but detectable with the as-PCR technique and therefore in 5xl0^"4%> and 0.1%) of cells. The results are in good agreement with SFHR gene repair frequencies achieved in pivotal SFHR experiments by others (Goncz et al., 1998; Kunzelmann et al, 1996). EXAMPLE 10 IVO AND IN VITRO CORRECTION OF THE mdx DYSTROPHIN GENE NONSENSE MUTATION BY SHORT FRAGMENT HOMOLOGOUS REPLACEMENT

Expression of Repaired dys Loci

RT PCR

After passage harvesting for DNA extraction, the remainders of cultures 1 through to 3 a were allowed to undergo myotube formation. After 7 days of differentiation, Cultures 1, la, 3, 3a and cultures 2 and 2a respectively, were pooled respectively to establish two composite repaired myotube sources, centrifuged and pellets frozen for RNA analysis. RNA extracted from these composite myotubes represented single and double SFHR applications respectively. The RNA obtained from these cultures was analyzed by RT PCR for dystrophin transcript as described earlier and shown in Figure 5. No dystrophin transcript was detected in these myotube cultures (Fig. 14A, lane 2/2a). Neither was there any dystrophin transcript detected in cultures subjected to sham lipofection (Fig 14 A, lane mdx-C) where no SFHR was performed (no Amplicon C). In RT PCR reactions performed on RNA extracted from mdx and wt muscle, dystrophin transcript was detected in amounts of RNA corresponding to that used in the myotube culture RNA (Fig 5A, lanes wt-m and mdx-m). RNA integrity in the muscle and myotube extractions was assessed by RT PCR amplification of Glyceraldehyde-3 -Phosphate Dehydrogenase (GAP-DH) transcript (equal quantities) from the wt-m and 2/2a myotube extracts (Fig 14B).

Both the muscle and myotube RNA could equally be used for RT PCR. The cultures containing the repaired genes were thus shown not to be expressing detectable levels of dystrophin transcript. B. Immunohistochemistry

Aliquots of the passaged cells were grown and allowed to undergo differentiation on chamber slides. Cell numbers diminished markedly during the differentiation process and differentiation beyond seven days left very few cells/tubes on the slide. This effect was considerably more marked in multiple SFHR application cultures, where again cultures were significantly depleted by 7 days of differentiation. Immunohistochemistry of the myotubes on these slides revealed no dystrophin expression that differed to untreated mdx myotubes.

EXAMPLE 11

IN VIVO AND IN VITRO CORRECTION OF THE mdx DYSTROPHIN GENE

NONSENSE MUTATION BY SHORT FRAGMENT HOMOLOGOUS

REPLACEMENT SFHR Gene Repair in mdx Mouse Tibialis Anterior

Three weeks after injection of the SFHR-wt and SFHR- vehicle cocktails into the right and left TAs of the 4 male mdx mice, the mice were sacrificed by cervical dislocation and TAs removed for immunohistochemistry and DNA screening. The frozen TAs were sectioned and immunohistochemically stained with anti-dystrophin antibody. As for in vitro SFHR, no differences were observed between amplicon and vehicle injected TAs in the numbers or extent of fibres staining positive for Dystrophin.

Following immunohistochemical examination, DNA was extracted from these tissues and showed that all four TAs injected with the DNA cocktail showed repair at the dys locus. In contrast, the DNA from contralateral TAs injected with saline cocktail lacked evidence of repair at the dys locus. Application of the PCR-RFLP technique to these tissues did not reveal sufficient DNA repair to visualise by this method. This result suggests that in vivo SFHR repair of the dys locus occurred at a frequency somewhere between 5xl0^"4% and 0.1%) of the regenerating cells in the injected mdx TAs. The relatively low frequency of gene repair achieved in vivo, whilst promising, underlies the lack of detectable dystrophin expression by immunohistochemistry both for in vivo applications presented here, as well as in the in vitro application of SFHR.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

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Primer Name 51 ] Primer Seαuence 51 Direction Purpose

3 6 9 12 15 18 21 . 24 27 30

A (Dys In22 S-01) <400>1 66 CAC TAT GAT TAA ATG CTT GAT ATT GAG 92 Sense Fragment A

B (Dys M22 S-02) <400>2 177 GTT GAT TCT AAA AAT CCC ATG TTG 200 Sense asPCR

CO C (Dys In22 S-03) <400>3 275 GTT TCA CTG TAG GTA AGT AAA TGT ATC AC 303 Sense Fragment C c m D (Dys In23 AS-01) <400>4 875 GGC TTT TGA TAT CAT CAA TAT CTT TGA AGG 846 Anti-Sense Fragments

CO

H A&C

H C H Dys-wt AS-01 <400>5 598 GTC ACT CAG ATA GTT GAA GCC ATT TTG 572 Anti-Sense asPCR m

CO Dys- re S-01* <400>6 546 CTC TGC AAA GTT CTT TGA AAG AGT AA 571 Sense PCR-RFLP

I m m Dys Ex23 AS-01 <400>7 656 CTG ACA GAT ATT TCT GGC ATA TTT C 631 Anti-Sense PCR-RFLP

H c3603-AS*** <400>8 3581 CTG GAT GCA AAC TCA AGT TCA GC 3603 Anti-Sense RT & RT PCR

*J

C I- C2801-S** <400>9 2800 CAC CCT ATC AGA GCC AAC AGC 2821 Anti-Sense RT PCR m t σ>

73 Note: * Sequence modifications from native wt sequence are indicated by Bold and Underlined text. O

5 C **Indicates numbering according to dystrophin cDNA Sequence Identity Numbers (SEQ ID NOs.) for the nucleotide and amino acid sequences referred to in the specification are defined following the bibliography. A summary of the SEQ ID NOs. is provided before the Examples.

TABLE 1. Oligonucleotides for SFHR of the dys Gene in mdx Mouse Myoblasts. The sequences of all oligonucleotides, their location with respect to the sequence mapped in Fig. 1 and their purpose in this study are shown. Nucleotide modifications in Oligonucleotides that vary from the native sequence are shown in underlined boldface

Claims

CLAIMS:

1. A method for treating a condition in an individual, which condition is attributable at least in part to the expression of a defective gene, said method comprising administering to said individual an effective number of autologous genetically altered cells, wherein said cells in their non-altered form are characterised by the subject defective gene, said genetic alteration comprising the introduction of at least one modification to said defective gene wherein said genetically altered cells are characterised by the capacity to produce an expression product or derivative, homologue, analogue, mutant or mimetic thereof which exhibits improved functional activity relative to the expression product produced by said defective gene.

2. The method according to claim 1 wherein said gene is the dystrophin gene.

3. The method according to claim 1 or 2 wherein said genetic alteration comprises the insertion of a nucleic acid molecule or derivative or analogue thereof, which molecule comprises at least one nucleotide.

4. The method according to claim 3 wherein said defect is an unwanted nucleic acid deletion and said genetic alteration comprises the insertion of a nucleic acid molecule or derivative or analogue thereof at the deletion interface.

5. The method according to claim 3 wherein said defect is a mutated nucleotide and said genetic alteration comprises the insertion of a nucleic acid molecule or derivative or analogue thereof to the genomic sequence.

6. The method according to any one of claims 3-5 wherein said genetic alteration is achieved utilising the technique of homologous recombination or chimeroplasty.

7. The method according to claim 6 wherein said homologous recombination is small fragment homologous recombination.

8. The method according to any one of claims 1-7 wherein said cells are stem cells.

9. The method according to claim 2 wherein said defective gene is characterised by a frame shift, nonsense mutation or in frame deletion of the dystrophin gene region encoding the cysteine-rich and/or C-terminal domains of dystrophin.

10. The method according to claim 9 wherein said genetic alteration comprises the introduction of at least one modification to one or more of exons 62-72 of said dystrophin encoding gene.

11. The method according to claim 2 wherein said defective gene is characterised by a nucleotide deletion or point mutation of the dystrophin gene region encoding the helical rod domain of dystrophin.

12. The method according to any one of claims 9-11 wherein said genetic alteration is achieved utilising the technique of homologous recombination or chimeroplasty.

13. The method according to claim 12 wherein said homologous recombination is small fragment homologous recombination.

14. The method according to any one of claims 9-13 wherein said condition is dystrophin deficiency.

15. The method according to claim 14 wherein said cells are myocytes, precursor muscle cells or stem cells.

16. The method according to claim 15 wherein said precursor muscle cells are myoblasts.

17. A method for the therapeutic and/or prophylactic treatment of a disease condition, in an individual which disease condition is characterised by the expression of a defective gene, said method comprising administering to said individual an effective number of genetically altered autologous cells, which cells in their non- altered form are characterised by the subject defective gene, said genetic alteration comprising the introduction of at least one modification to said defective gene wherein said genetically altered cells are characterised by the capacity to produce an expression product or derivative, homologue, analogue, mutant or mimetic thereof which exhibits improved functional activity relative to the expression product produced by said defective gene.

18. The method according to claim 17 wherein said gene is the dystrophin gene.

19. The method according to claim 18 wherein said condition is dystrophin deficiency.

20. The method according to any one of claims 17-19 wherein said genetic alteration comprises the insertion of a nucleic acid molecule or derivative or analogue thereof, which molecule comprises at least one nucleotide.

21. The method according to claim 20 wherein said defect is an unwanted nucleic acid deletion and said genetic alteration comprises the insertion of a nucleic acid molecule or derivative or analogue thereof at the deletion interface.

22. The method according to claim 20 wherein said defect is a mutated nucleotide and said genetic alteration comprises the insertion of a nucleic acid molecule or derivative or analogue thereof to the genomic sequence.

23. The method according to any one of claims 20-22 wherein said genetic alteration is achieved utilising the technique of homologous recombination or chimeroplasty.

24. The method according to claim 23 wherein said homologous recombination is small fragment homologous recombination.

25. The method according to any one of claims 17-24 wherein said cells are stem cells.

26. The method according to claims 18-19 wherein said defective gene is characterised by a frame shift, nonsense mutation or in frame deletion of the dystrophin gene region encoding the cysteine-rich and/or C-terminal domains of dystrophin.

27. The method according to claim 26 wherein said genetic alteration comprises the introduction of at least one modification to one or more of exons 62-72 of said dystrophin encoding gene.

28. The method according to claims 18-19 wherein said defective gene is characterised by a nucleotide deletion or point mutation of the dystrophin gene region encoding the helical rod domain of dystrophin.

29. The method according to any one of claims 18 or 19 wherein said condition is Duchenne Muscular Dystrophy.

30. The method according to claim 29 wherein said genetic alteration is a modification to the dystrophin gene region encoding the cysteine-rich domain and/or the C- terminal domain.

31. The method according to claim 29 wherein said genetic alteration is a modification to one or more of exons 62-72 and/or 74.

32. The method according to any one of claims 18 or 19 wherein said condition is Becker's Muscular Dystrophy.

33. The method according to claim 32 wherein said genetic alteration is a modification to the dystrophin gene region encoding the helical rod domain.

34. The method according to any one of claims 18-33 wherein said genetic alteration is achieved utilising the technique of homologous recombination or chimeroplasty.

35. The method according to claim 34 wherein said homologous recombination is small fragment homologous recombination.

36. The method according to any one of claims 18-19 or 26-35 wherein said cells are myocytes, precursor muscle cells or stem cells.

37. The method according to claim 36 wherein said precursor muscle cells are myoblasts.

38. Use of genetically altered cells, which cells in their non-altered form are characterised by a defective gene, in the manufacture of a medicament for the treatment of a condition characterised by the expression of said defective gene, wherein said genetic alteration comprises the introduction of at least one modification to said defective gene wherein said genetically altered cells are characterised by the capacity to produce an expression product or derivative, homologue, analogue, mutant or mimetic thereof which exhibits improved functional activity relative to the expression product produced by said defective gene.

39. Use according to claim 38 wherein said gene is the dystrophin gene.

40. Use according to claims 38 or 39 wherein said genetic alteration comprises the insertion of a nucleic acid molecule or derivative or analogue thereof, which molecule comprises at least nucleotide.

41. Use according to claim 40 wherein said defect is an unwanted nucleic acid deletion and said genetic alteration comprises the insertion of a nucleic acid molecule or derivative or analogue thereof at the deletion interface.

42. Use according to claim 40 wherein said defect is a mutated nucleotide and said genetic alteration comprises the insertion of a nucleic acid molecule or derivative or analogue thereof to the genomic sequence.

43. Use according to claims 40-42 wherein said genetic alteration is achieved utilising the technique of homologous recombination or chimeroplasty.

44. Use according to claim 43 wherein said homologous recombination is small fragment homologous recombination.

45. Use according to claims 38-44 wherein said cells are stem cells.

46. Use according to claim 39 wherein said defective gene is characterised by a frame shift, nonsense mutation or in frame deletion of the dystrophin gene region encoding the cysteine-rich and/or C-terminal domains of dystrophin.

47. Use according to claim 46 wherein said genetic alteration comprises the introduction of at least one modification to one or more of exons 62-72 of said dystrophin encoding gene.

48. Use according to claim 39 wherein said defective gene is characterised by a nucleotide deletion or point mutation of the dystrophin gene region encoding the helical rod domain of dystrophin.

49. Use according to claims 38-48 wherein said genetic alteration is achieved utilising the technique of homologous recombination or chimeroplasty.

50. Use according to claim 49 wherein said homologous recombination is small fragment homologous recombination.

51. Use according to claims 46-48 wherein said condition is dystrophin deficiency.

52. Use according to claim 51 wherein said cells are myocytes, precursor muscle cells or stem cells.

53. Use according to claim 52 wherein said precursor muscle cells are myoblasts.

54. Use according to claim 39 wherein said condition is dystrophin deficiency.

55. Use according to claim 54 wherein said condition is Duchenne Muscular Dystrophy.

56. Use according to claim 55 wherein said genetic alteration is a modification to the dystrophin gene region encoding the cysteine-rich domain and/or the C-terminal domain.

57. Use according to claim 55 wherein said genetic alteration is a modification to one or more of exons 62-72 and/or 74.

58. Use according to claim 54 wherein said condition is Becker's Muscular Dystrophy.

59. Use according to claim 58 wherein said genetic alteration is a modification to the dystrophin gene region encoding the helical rod domain.

60. Use according to any one of claims 56, 57 or 59 wherein said genetic alteration is achieved utilising the technique of homologous recombination or chimeroplasty.

61. Use according to claim 60 wherein said homologous recombination is small fragment homologous recombination.

62. Use according to any one of claims 54-61 wherein said cells are stem cells, myocytes or precursor muscle cells.

63. Use according to claim 62 wherein said precursor muscle cells are myoblasts.

64. Genetically altered cells as defined in accordance with any one of claims 1-37 when used in accordance with the method of any one of claims 1-37.