Enhanced delivery of peptide-morpholino oligonucleotides with a small molecule to correct splicing defects in the lung.

Pulmonary diseases offer many targets for oligonucleotide therapeutics. However, effective delivery of oligonucleotides to the lung is challenging. For example, splicing mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) affect a significant cohort of Cystic Fibrosis (CF) patients. These individuals could potentially benefit from treatment with splice switching oligonucleotides (SSOs) that can modulate splicing of CFTR and restore its activity. However, previous studies in cell culture used oligonucleotide transfection methods that cannot be safely translated in vivo. In this report, we demonstrate effective correction of a splicing mutation in the lung of a mouse model using SSOs. Moreover, we also demonstrate effective correction of a CFTR splicing mutation in a pre-clinical CF patient-derived cell model. We utilized a highly effective delivery strategy for oligonucleotides by combining peptide-morpholino (PPMO) SSOs with small molecules termed OECs. PPMOs distribute broadly into the lung and other tissues while OECs potentiate the effects of oligonucleotides by releasing them from endosomal entrapment. The combined PPMO plus OEC approach proved to be effective both in CF patient cells and in vivo in the mouse lung and thus may offer a path to the development of novel therapeutics for splicing mutations in CF and other lung diseases.

Effects of systemic multiexon skipping with peptide-conjugated morpholinos in the heart of a dog model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a lethal genetic disorder caused by an absence of the dystrophin protein in bodywide muscles, including the heart. Cardiomyopathy is a leading cause of death in DMD. Exon skipping via synthetic phosphorodiamidate morpholino oligomers (PMOs) represents one of the most promising therapeutic options, yet PMOs have shown very little efficacy in cardiac muscle. To increase therapeutic potency in cardiac muscle, we tested a next-generation morpholino: arginine-rich, cell-penetrating peptide-conjugated PMOs (PPMOs) in the canine X-linked muscular dystrophy in Japan (CXMDJ) dog model of DMD. A PPMO cocktail designed to skip dystrophin exons 6 and 8 was injected intramuscularly, intracoronarily, or intravenously into CXMDJ dogs. Intravenous injections with PPMOs restored dystrophin expression in the myocardium and cardiac Purkinje fibers, as well as skeletal muscles. Vacuole degeneration of cardiac Purkinje fibers, as seen in DMD patients, was ameliorated in PPMO-treated dogs. Although symptoms and functions in skeletal muscle were not ameliorated by i.v. treatment, electrocardiogram abnormalities (increased Q-amplitude and Q/R ratio) were improved in CXMDJ dogs after intracoronary or i.v. administration. No obvious evidence of toxicity was found in blood tests throughout the monitoring period of one or four systemic treatments with the PPMO cocktail (12 mg/kg/injection). The present study reports the rescue of dystrophin expression and recovery of the conduction system in the heart of dystrophic dogs by PPMO-mediated multiexon skipping. We demonstrate that rescued dystrophin expression in the Purkinje fibers leads to the improvement/prevention of cardiac conduction abnormalities in the dystrophic heart.

Engineered U7 snRNA mediates sustained splicing correction in erythroid cells from ß-thalassemia/HbE patients.

Repair of a splicing defect of ß-globin pre-mRNA harboring hemoglobin E (HbE) mutation was successfully accomplished in erythroid cells from patients with ß-thalassemia/HbE disorder by a synthetic splice-switching oligonucleotide (SSO). However, its application is limited by short-term effectiveness and requirement of lifelong periodic administration of SSO, especially for chronic diseases like thalassemias. Here, we engineered lentiviral vectors that stably express U7 small nuclear RNA (U7 snRNA) carrying the splice-switching sequence of the SSO that restores correct splicing of ßE-globin pre-mRNA and achieves a long-term therapeutic effect. Using a two-step tiling approach, we systematically screened U7 snRNAs carrying splice-switching SSO sequences targeted to the cryptic 5' splice site created by HbE mutation. We tested this approach and identified the most responsive element for mediating splicing correction in engineered U7 snRNAs in HeLa-ßE cell model cell line. Remarkably, the U7 snRNA lentiviral vector (U7 ßE4+1) targeted to this region effectively restored the correctly-spliced ßE-globin mRNA for at least 5 months. Moreover, the effects of the U7 ßE4+1 snRNA lentiviral vector were also evident as upregulation of the correctly-spliced ßE-globin mRNA in erythroid progenitor cells from ß-thalassemia/HbE patients treated with the vector, which led to improvements of pathologies in erythroid progenitor cells from thalassemia patients. These results suggest that the splicing correction of ßE-globin pre-mRNA by the engineered U7 snRNA lentiviral vector provides a promising, long-term treatment for ß-thalassemia/HbE.

Oligonucleotide therapeutics in cancer.

Alterations in pre-mRNA splicing can have profound effects on gene expression and lead to cellular transformation. Oligonucleotide therapeutics are drugs that manipulate gene expression and improve the disease state. Antisense oligonucleotides hybridize with a target mRNA to downregulate gene expression via an RNase H-dependent mechanism. Additionally, RNase H-independent splice switching oligonucleotides (SSO) modulate alternative or aberrant splicing, to favor the therapeutically relevant splicing product. This chapter summarizes the progress made in the application of these oligonucleotide drugs in the treatment of cancer.

Exon skipping and dystrophin restoration in patients with Duchenne muscular dystrophy after systemic phosphorodiamidate morpholino oligomer treatment: an open-label, phase 2, dose-escalation study.

BACKGROUND: We report clinical safety and biochemical efficacy from a dose-ranging study of intravenously administered AVI-4658 phosphorodiamidate morpholino oligomer (PMO) in patients with Duchenne muscular dystrophy. METHOD: We undertook an open-label, phase 2, dose-escalation study (0·5, 1·0, 2·0, 4·0, 10·0, and 20·0 mg/kg bodyweight) in ambulant patients with Duchenne muscular dystrophy aged 5-15 years with amenable deletions in DMD. Participants had a muscle biopsy before starting treatment and after 12 weekly intravenous infusions of AVI-4658. The primary study objective was to assess safety and tolerability of AVI-4658. The secondary objectives were pharmacokinetic properties and the ability of AVI-4658 to induce exon 51 skipping and dystrophin restoration by RT-PCR, immunohistochemistry, and immunoblotting. The study is registered, number NCT00844597. FINDINGS: 19 patients took part in the study. AVI-4658 was well tolerated with no drug-related serious adverse events. AVI-4658 induced exon 51 skipping in all cohorts and new dystrophin protein expression in a significant dose-dependent (p=0·0203), but variable, manner in boys from cohort 3 (dose 2 mg/kg) onwards. Seven patients responded to treatment, in whom mean dystrophin fluorescence intensity increased from 8·9% (95% CI 7·1-10·6) to 16·4% (10·8-22·0) of normal control after treatment (p=0·0287). The three patients with the greatest responses to treatment had 21%, 15%, and 55% dystrophin-positive fibres after treatment and these findings were confirmed with western blot, which showed an increase after treatment of protein levels from 2% to 18%, from 0·9% to 17%, and from 0% to 7·7% of normal muscle, respectively. The dystrophin-associated proteins α-sarcoglycan and neuronal nitric oxide synthase were also restored at the sarcolemma. Analysis of the inflammatory infiltrate indicated a reduction of cytotoxic T cells in the post-treatment muscle biopsies in the two high-dose cohorts. INTERPRETATION: The safety and biochemical efficacy that we present show the potential of AVI-4658 to become a disease-modifying drug for Duchenne muscular dystrophy. FUNDING: UK Medical Research Council; AVI BioPharma.

Anti-tumor activity of splice-switching oligonucleotides.

Alternative splicing has emerged as an important target for molecular therapies. Splice-switching oligonucleotides (SSOs) modulate alternative splicing by hybridizing to pre-mRNA sequences involved in splicing and blocking access to the transcript by splicing factors. Recently, the efficacy of SSOs has been established in various animal disease models; however, the application of SSOs against cancer targets has been hindered by poor in vivo delivery of antisense therapeutics to tumor cells. The apoptotic regulator Bcl-x is alternatively spliced to express anti-apoptotic Bcl-x(L) and pro-apoptotic Bcl-x(S). Bcl-x(L) is upregulated in many cancers and is associated with chemoresistance, distinguishing it as an important target for cancer therapy. We previously showed that redirection of Bcl-x pre-mRNA splicing from Bcl-x(L) to -x(S) induced apoptosis in breast and prostate cancer cells. In this study, the effect of SSO-induced Bcl-x splice-switching on metastatic melanoma was assessed in cell culture and B16F10 tumor xenografts. SSOs were delivered in vivo using lipid nanoparticles. Administration of nanoparticle with Bcl-x SSO resulted in modification of Bcl-x pre-mRNA splicing in lung metastases and reduced tumor load, while nanoparticle alone or formulated with a control SSO had no effect. Our findings demonstrate in vivo anti-tumor activity of SSOs that modulate Bcl-x pre-mRNA splicing.

RNA repair restores hemoglobin expression in IVS2-654 thalassemic mice.

Repair of beta-globin pre-mRNA rendered defective by a thalassemia-causing splicing mutation, IVS2-654, in intron 2 of the human beta-globin gene was accomplished in vivo in a mouse model of IVS2-654 thalassemia. This was effected by a systemically delivered splice-switching oligonucleotide (SSO), a morpholino oligomer conjugated to an arginine-rich peptide. The SSO blocked the aberrant splice site in the targeted pre-mRNA and forced the splicing machinery to reselect existing correct splice sites. Repaired beta-globin mRNA restored significant amounts of hemoglobin in the peripheral blood of the IVS2-654 mouse, improving the number and quality of erythroid cells.

Mismatched single stranded antisense oligonucleotides can induce efficient dystrophin splice switching.

BACKGROUND: Antisense oligomer induced exon skipping aims to reduce the severity of Duchenne muscular dystrophy by redirecting splicing during pre-RNA processing such that the causative mutation is by-passed and a shorter but partially functional Becker muscular dystrophy-like dystrophin isoform is produced. Normal exons are generally targeted to restore the dystrophin reading frame however, an appreciable subset of dystrophin mutations are intra-exonic and therefore have the potential to compromise oligomer efficiency, necessitating personalised oligomer design for some patients. Although antisense oligomers are easily personalised, it remains unclear whether all patient polymorphisms within antisense oligomer target sequences will require the costly process of producing and validating patient specific compounds. METHODS: Here we report preclinical testing of a panel of splice switching antisense oligomers, designed to excise exon 25 from the dystrophin transcript, in normal and dystrophic patient cells. These patient cells harbour a single base insertion in exon 25 that lies within the target sequence of an oligomer shown to be effective at removing exon 25. RESULTS: It was anticipated that such a mutation would compromise oligomer binding and efficiency. However, we show that, despite the mismatch an oligomer, designed and optimised to excise exon 25 from the normal dystrophin mRNA, removes the mutated exon 25 more efficiently than the mutation-specific oligomer. CONCLUSION: This raises the possibility that mismatched AOs could still be therapeutically applicable in some cases, negating the necessity to produce patient-specific compounds.

Prevention of dystrophic pathology in severely affected dystrophin/utrophin-deficient mice by morpholino-oligomer-mediated exon-skipping.

Duchenne muscular dystrophy (DMD) is a severe neuromuscular disorder caused by mutations in the dystrophin gene that result in the absence of functional protein. Antisense-mediated exon-skipping is one of the most promising approaches for the treatment of DMD because of its capacity to correct the reading frame and restore dystrophin expression, which has been demonstrated in vitro and in vivo. In particular, peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) have recently been shown to induce widespread high levels of dystrophin expression in the mdx mouse model. Here, we report the efficiency of the PPMO-mediated exon-skipping approach in the utrophin/dystrophin double-knockout mouse (dKO) mouse, which is a much more severe and progressive mouse model of DMD. Repeated intraperitoneal (i.p.) injections of a PPMO targeted to exon 23 of dystrophin pre-mRNA in dKO mice induce a near-normal level of dystrophin expression in all muscles examined, except for the cardiac muscle, resulting in a considerable improvement of their muscle function and dystrophic pathology. These findings suggest great potential for PPMOs in systemic treatment of the DMD phenotype.

Targeted correction of a thalassemia-associated beta-globin mutation induced by pseudo-complementary peptide nucleic acids.

Beta-thalassemia is a genetic disorder caused by mutations in the beta-globin gene. Triplex-forming oligonucleotides and triplex-forming peptide nucleic acids (PNAs) have been shown to stimulate recombination in mammalian cells via site-specific binding and creation of altered helical structures that provoke DNA repair. However, the use of these molecules for gene targeting requires homopurine tracts to facilitate triple helix formation. Alternatively, to achieve binding to mixed-sequence target sites for the induced gene correction, we have used pseudo-complementary PNAs (pcPNAs). Due to steric hindrance, pcPNAs are unable to form pcPNA-pcPNA duplexes but can bind to complementary DNA sequences via double duplex-invasion complexes. We demonstrate here that pcPNAs, when co-transfected with donor DNA fragments, can promote single base pair modification at the start of the second intron of the beta-globin gene. This was detected by the restoration of proper splicing of transcripts produced from a green fluorescent protein-beta globin fusion gene. We also demonstrate that pcPNAs are effective in stimulating recombination in human fibroblast cells in a manner dependent on the nucleotide excision repair factor, XPA. These results suggest that pcPNAs can be effective tools to induce heritable, site-specific modification of disease-related genes in human cells without purine sequence restriction.

Correction of a splice-site mutation in the beta-globin gene stimulated by triplex-forming peptide nucleic acids.

Splice-site mutations in the beta-globin gene can lead to aberrant transcripts and decreased functional beta-globin, causing beta-thalassemia. Triplex-forming DNA oligonucleotides (TFOs) and peptide nucleic acids (PNAs) have been shown to stimulate recombination in reporter gene loci in mammalian cells via site-specific binding and creation of altered helical structures that provoke DNA repair. We have designed a series of triplex-forming PNAs that can specifically bind to sequences in the human beta-globin gene. We demonstrate here that these PNAs, when cotransfected with recombinatory donor DNA fragments, can promote single base-pair modification at the start of the second intron of the beta-globin gene, the site of a common thalassemia-associated mutation. This single base pair change was detected by the restoration of proper splicing of transcripts produced from a green fluorescent protein-beta-globin fusion gene. The ability of these PNAs to induce recombination was dependent on dose, sequence, cell-cycle stage, and the presence of a homologous donor DNA molecule. Enhanced recombination, with frequencies up to 0.4%, was observed with use of the lysomotropic agent chloroquine. Finally, we demonstrate that these PNAs were effective in stimulating the modification of the endogenous beta-globin locus in human cells, including primary hematopoietic progenitor cells. This work suggests that PNAs can be effective tools to induce heritable, site-specific modification of disease-related genes in human cells.

Engineered U7 Small Nuclear RNA Restores Correct ß-Globin Pre-mRNA Splicing in Mouse ßIVS2-654-Thalassemic Erythroid Progenitor Cells.

Restoration of correct splicing of ßIVS2-654-globin pre-mRNA was previously accomplished in erythroid cells from ß-thalassemia/HbE patients by an engineered U7 small nuclear RNA (snRNA) that carried a sequence targeted to the cryptic branch point and an exonic splicing enhancer, U7.BP+623 snRNA. In this study, this approach was tested in thalassemic mice carrying the ßIVS2-654 mutation. While correction of ßIVS2-654 pre-mRNA splicing was achieved in erythroid progenitors transduced with a lentiviral vector carrying the U7.BP+623 snRNA, a high level of truncated U7.BP+623 snRNA was also observed. The discrepancy of processing of the modified U7 snRNA in human and mouse constructs hamper the evaluation of pathologic improvement in mouse model.

A targeted antisense therapeutic approach for Hutchinson-Gilford progeria syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare accelerated aging disorder characterized by premature death from myocardial infarction or stroke. It is caused by de novo single-nucleotide mutations in the LMNA gene that activate a cryptic splice donor site, resulting in the production of a toxic form of lamin A, which is termed progerin. Here we present a potential genetic therapeutic strategy that utilizes antisense peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) to block pathogenic splicing of mutant transcripts. Of several candidates, PPMO SRP-2001 provided the most significant decrease in progerin transcripts in patient fibroblasts. Intravenous delivery of SRP-2001 to a transgenic mouse model of HGPS produced significant reduction of progerin transcripts in the aorta, a particularly critical target tissue in HGPS. Long-term continuous treatment with SRP-2001 yielded a 61.6% increase in lifespan and rescue of vascular smooth muscle cell loss in large arteries. These results provide a rationale for proceeding to human trials.

Modification of HER2 pre-mRNA alternative splicing and its effects on breast cancer cells.

The oncogene HER2 is overexpressed in a variety of human tumors, providing a target for anti-cancer molecular therapies. Here, we employed a 2'-O-methoxyethyl (MOE) splice switching oligonucleotide, SSO111, to induce skipping of exon 15 in HER2 pre-mRNA, leading to significant downregulation of full-length HER2 mRNA, and simultaneous upregulation of Delta15HER2 mRNA. SSO111 treatment of SK-BR-3 cells, which highly overexpress HER2, led to inhibition of cell proliferation and induction of apoptosis. The novel Delta15HER2 mRNA encodes a soluble, secreted form of the receptor. Treating SK-BR-3 cells with exogenous Delta15HER2 protein reduced membrane-bound HER2 and decreased HER3 transphosphorylation. Delta15HER2 protein thus has similar activity to an autoinhibitory, natural splice variant of HER2, Herstatin, and to the breast cancer drug Herceptin. Both SSO111 and Delta15HER2 may be potential candidates for the development of novel HER2-targeted cancer therapeutics.

An endogenous TNF-alpha antagonist induced by splice-switching oligonucleotides reduces inflammation in hepatitis and arthritis mouse models.

Tumor necrosis factor-alpha (TNF-alpha) is a key mediator of inflammatory diseases, including rheumatoid arthritis (RA), and anti-TNF-alpha drugs such as etanercept are effective treatments. Splice-switching oligonucleotides (SSOs) are a new class of drugs designed to induce therapeutically favorable splice variants of targeted genes. In this work, we used locked nucleic acid (LNA)-based SSOs to modulate splicing of TNF receptor 2 (TNFR2) pre-mRNA. The SSO induced skipping of TNFR2 exon 7, which codes the transmembrane domain (TM), switching endogenous expression from the membrane-bound, functional form to a soluble, secreted form (Delta7TNFR2). This decoy receptor protein accumulated in the circulation of treated mice, antagonized TNF-alpha, and altered disease in two mouse models: TNF-alpha-induced hepatitis and collagen-induced arthritis (CIA). This is the first report of upregulation of the endogenous, circulating TNF-alpha antagonist by oligonucleotide-induced splicing modulation.

Sustained dystrophin expression induced by peptide-conjugated morpholino oligomers in the muscles of mdx mice.

Cell-penetrating peptides (CPPs), containing arginine (R), 6-aminohexanoic acid (X), and/or beta-alanine (B) conjugated to phosphorodiamidate morpholino oligomers (PMOs), enhance their delivery in cell culture. In this study, the potency, functional biodistribution, and toxicity of these conjugates were evaluated in vivo, in EGFP-654 transgenic mice that ubiquitously express the aberrantly spliced EGFP-654 pre-mRNA reporter. Correct splicing and enhanced green fluorescence protein (EGFP) upregulation serve as a positive readout for peptide-PMO (PPMO) entry into cells and access to EGFP-654 pre-mRNA in the nucleus. Intraperitoneal injections of a series of PPMOs, A-N (12 mg/kg), administered once a day for four successive days resulted in splicing correction in numerous tissues. PPMO-B was highly potent in the heart, diaphragm, and quadriceps, which are key muscles in the treatment of Duchenne muscular dystrophy. We therefore investigated PPMO M23D-B, designed to force skipping of stop-codon containing dystrophin exon 23, in an mdx mouse model of the disease. Systemic delivery of M23D-B yielded persistent exon 23 skipping, yielding high and sustained dystrophin protein expression in body-wide muscles, including cardiac muscle, without detectable toxicity. The rescued dystrophin reduced serum creatinine kinase to near-wild-type levels, indicating improvement in muscle integrity. This is the first report of oligonucleotide-mediated exon skipping and dystrophin protein induction in the heart of treated animals.

An Endogenous TNF-α Antagonist Induced by Splice-switching Oligonucleotides Reduces Inflammation in Hepatitis and Arthritis Mouse Models.

Tumor necrosis factor-α (TNF-α) is a key mediator of inflammatory diseases, including rheumatoid arthritis (RA), and anti-TNF-α drugs such as etanercept are effective treatments. Splice-switching oligonucleotides (SSOs) are a new class of drugs designed to induce therapeutically favorable splice variants of targeted genes. In this work, we used locked nucleic acid (LNA)-based SSOs to modulate splicing of TNF receptor 2 (TNFR2) pre-mRNA. The SSO induced skipping of TNFR2 exon 7, which codes the transmembrane domain (TM), switching endogenous expression from the membrane-bound, functional form to a soluble, secreted form (Δ7TNFR2). This decoy receptor protein accumulated in the circulation of treated mice, antagonized TNF-α, and altered disease in two mouse models: TNF-α-induced hepatitis and collagen-induced arthritis (CIA). This is the first report of upregulation of the endogenous, circulating TNF-α antagonist by oligonucleotide-induced splicing modulation.

Restoration of correct ßIVS2-654-globin mRNA splicing and HbA production by engineered U7 snRNA in ß-thalassaemia/HbE erythroid cells.

A cytosine to thymine mutation at nucleotide 654 of human ß-globin intron 2 (ßIVS2-654) is one of the most common mutations causing ß-thalassaemia in Chinese and Southeast Asians. This mutation results in aberrant ß-globin pre-mRNA splicing and prevents synthesis of ß-globin protein. Splicing correction using synthetic splice-switching oligonucleotides (SSOs) has been shown to restore expression of the ß-globin protein, but to maintain therapeutically relevant levels of ß-globin it would require lifelong administration. Here, we demonstrate long-term splicing correction using U7 snRNA lentiviral vectors engineered to target several pre-mRNA splicing elements on the ßIVS2-654-globin pre-mRNA such as cryptic 3' splice site, aberrant 5' splice site, cryptic branch point and an exonic splicing enhancer. A double-target engineered U7 snRNAs targeted to the cryptic branch point and an exonic splicing enhancer, U7.BP + 623, was the most effective in a model cell line, HeLa IVS2-654. Moreover, the therapeutic potential of the vector was demonstrated in erythroid progenitor cells derived from ßIVS2-654-thalassaemia/HbE patients, which showed restoration of correctly spliced ß-globin mRNA and led to haemoglobin A synthesis, and consequently improved thalassaemic erythroid cell pathology. These results demonstrate proof of concept of using the engineered U7 snRNA lentiviral vector for treatment of ß-thalassaemia.

Therapeutic potential of antisense oligonucleotides as modulators of alternative splicing.

An estimated 60% of all human genes undergo alternative splicing, a highly regulated process that produces splice variants with different functions. Such variants have been linked to a variety of cancers, and genetic diseases such as thalassemia and cystic fibrosis. This Perspective describes a promising approach to RNA repair based on the use of antisense oligonucleotides to modulate alternative splicing and engender the production of therapeutic gene products.