Matthew G. Dunckley

Bifunctional antisense oligonucleotides provide a trans-acting splicing enhancer that stimulates SMN2 gene expression in patient fibroblasts

The multiplicity of proteins compared with genes in mammals owes much to alternative splicing. Splicing signals are so subtle and complex that small perturbations may allow the production of new mRNA variants. However, the flexibility of splicing can also be a liability, and several genetic diseases result from single-base changes that cause exons to be skipped during splicing. Conventional oligonucleotide strategies can block reactions but cannot restore splicing. We describe here a method by which the use of a defective exon was restored. Spinal muscular atrophy (SMA) results from mutations of the Survival Motor Neuron (SMN) gene. Mutations of SMN1 cause SMA, whereas SMN2 acts as a modifying gene. The two genes undergo alternative splicing with SMN1, producing an abundance of full-length mRNA transcripts, whereas SMN2 predominantly produces exon 7-deleted transcripts. This discrepancy is because of a single nucleotide difference in SMN2 exon 7, which disrupts an exonic splicing enhancer containing an SF2/ASF binding site. We have designed oligoribonucleotides that are complementary to exon 7 and contain exonic splicing enhancer motifs to provide trans-acting enhancers. These tailed oligoribonucleotides increased SMN2 exon 7 splicing in vitro and rescued the incorporation of SMN2 exon 7 in SMA patient fibroblasts. This treatment also resulted in the partial restoration of gems, intranuclear structures containing SMN protein that are severely reduced in patients with SMA. The use of tailed antisense oligonucleotides to recruit positively acting factors to stimulate a splicing reaction may have therapeutic applications for genetic disorders, such as SMA, in which splicing patterns are altered.

Retroviral‐mediated transfer of a dystrophin minigene into mdx mouse myoblasts in vitro

We have demonstrated expression of a 6.3 kb Becker muscular dystrophy (BMD) human dystrophin cDNA following retroviral‐mediated transduction of cultured myoblasts from the dystrophin‐deficient mdx mouse. The truncated dystrophin protein was localised to the sarcolemma of differentiated myotubes by antibodies against the C‐terminus of the molecule, and produced an identical immunostaining pattern to that observed in control myotubes expressing normal endogenous dystrophin. These results indicate that retroviral‐mediated gene transfer may be useful for experimental in vivo studies on the complementation of dystrophin gene mutations.

Lipofection of cultured mouse muscle cells : A direct comparison of lipofectamine and DOSPER

Cationic lipid–DNA complexes (lipoplexes) have been widely used as gene transfer vectors which avoid the adverse immunogenicity and potential for viraemia of viral vectors. With the long-term aim of gene transfer into skeletal muscle in vivo, we describe a direct in vitro comparison of two commercially available cationic lipid formulations, Lipofectamine and DOSPER. Optimisation of transfection was performed in the C2C12 mouse muscle cell line, before further studies in primary mouse myoblasts and C2C12 myotubes. Reporter gene constructs expressing either E. coli β-galactosidase or green fluorescent protein (GFP) were used in order to evaluate transfection efficiency by histochemical staining or FACS analysis, respectively. Both lipid formulations were able to promote efficient, reproducible gene transfer in C2C12 cells, and to transfect primary mouse myoblast cultures successfully. However, DOSPER exhibited the important advantage of being able to transfect cells in the presence of serum of both bovine and murine origin. This feature allowed increased cell survival during in vitro transfections, and may be advantageous for direct in vivo gene transfer efficacy.

Modulation of Splicing in the DMD Gene by Antisense Oligoribonucleotides

Abstract Splicing of the DMD gene pre-mRNA is being examined as a model system to study the skipping of mutant exons, especially where disrupted translational reading frames are restored. Naturally-occurring examples and induced exon skipping by specific synthetic RNA oligonucleotides are under investigation.

Efficient coexpression and secretion of anti-atherogenic human apolipoprotein AI and lecithin-cholesterol acyltransferase by cultured muscle cells using adeno-associated virus plasmid vectors

Plasma apolipoprotein AI (apoAI) and lecithin-cholesterol acyltransferase (LCAT) play important roles in reverse cholesterol transport, promoting the removal of excess cholesterol from peripheral cells and reducing formation of atherosclerotic lesions. Gene augmentation of either apoAI or LCAT, or both, are thus attractive targets for prevention or treatment of atherosclerosis. With the eventual aim of safe and efficient gene delivery to skeletal muscle, our chosen secretory platform for systemic delivery of anti-atherogenic proteins, we have constructed conventional and AAV-based plasmid vectors containing human apoAI or LCAT cDNAs; their efficacy was tested by lipoplex transfection of mouse C2C12 muscle cells or human 293 cells. The secretion of apoAI or LCAT by transduced cultures was two- to five-fold higher using AAV-based plasmid vectors than conventional plasmid vectors. Additionally, cells transfected with a bicistronic AAV-based vector containing an internal ribosome entry site (IRES) efficiently expressed both apoAI and LCAT simultaneously. Furthermore, AAV-based vector sequences were retained by host cells, whereas those of conventional plasmid vectors were lost. These studies indicate that ectopic overexpression of apoAI and LCAT in muscle tissue using AAV-based plasmid vectors might provide a feasible anti-atherogenic strategy in vivo.

Adenovirus-Based Targeting in Myoblasts Is Hampered by Nonhomologous Vector Integration

Chromosomal correction of dystrophin gene mutations is a most desirable therapeutic solution for Duchenne muscular dystrophy, as it allows production of the full-length dystrophin under the control of locus-specific promoters. Here we explored gene targeting in conditionally immortal mouse dystrophin-deficient myoblasts. We constructed an adenoviral vector for the correction of the mdx mutation, containing 6.0 kb of sequence homologous to the target locus (partial intron 21 through to exon 24 with the normal sequence of exon 23) and a neomycin expression cassette inserted in intron 23. Adenovirus-based gene targeting was previously reported to be beneficial in mouse embryonic stem cells, resulting in one targeted integration per three integration events. However, we found no targeted integration events among 144 stably transduced G418-resistant myoblast clones, reflecting efficient random integration of the adenoviral vector in myogenic cells. We found that mouse myoblasts are capable of integrating recombinant adenoviral DNA with an efficiency approaching 1%. Interestingly, dermal fibroblasts integrate adenoviral DNA up to 100 times less efficiently than myoblasts from the same mice. We also show that the efficiency of recombinant adenoviral DNA integration is influenced by preinfection cell density, possibly indicating the importance of cellular DNA replication for adenoviral integration.

Human dystrophin gene transfer: genetic correction of dystrophin deficiency

Somatic gene therapy, as generally conceived, involves reconstituting a biological function by adding a normal gene to somatic, i.e. non-germline, cells which are genetically deficient in that gene product (Friedmann, 1989). Single gene or Mendelian disorders which are recessive (autosomal or X-linked) are particularly attractive candidates for such a therapeutic approach since successful introduction of one normal gene copy would be expected to prevent the pathological phenotype. Moreover, as has been noted for many inborn errors of metabolism, even relatively low levels of residual function (5–10% of normal) may prevent the major clinical pathology associated with complete deficiency (Ledley, 1990). Thus, full restoration of physiological levels of deficient gene product, while desirable, may not be necessary for the clinical efficacy of any particular gene therapeutic strategy.

Retroviral-Mediated Gene Transfer and Duchenne Muscular Dystrophy

Over 70 human diseases have been named as potential beneficiaries of somatic cell gene therapy, ranging from inherited disorders, such as cystic fibrosis and forms of severe combined immune deficiency, to cancer and AIDS (Scarpa and Caskey, 1989). Intervention by ‘designer genes’ incorporated into patients’ own cells could replace absent or defective molecules, synthesize metabolic enzymes or drugs, or manipulate the body’s immune responses (Ledley, 1990; Miller, 1990a). The realisation that recombinant DNA technology, now familiar in a diagnostic context, can be applied to the correction of diagnosed diseases is leading to a renewed optimism in the clinic for treating conditions that have so far persistently defeated more conventional therapeutic strategies.