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`
`Molecules 2009, 14, 1304-1323; doi:10.3390/molecules14031304
`
`OPEN ACCESS
`
`molecules
`
`ISSN 1420-3049
`www.mdpi.com/journal/molecules
`
`Review
`Gene Knockdowns in Adult Animals: PPMOs and
`Vivo-Morpholinos
`
`Jon D. Moulton * and Shan Jiang
`
`Gene Tools, LLC, 1001 Summerton Way, Philomath, OR 97370 USA
`
`* Author to whom correspondence should be addressed; E-mail: jmoulton@gene-tools.com.
`
`Received: 2 March 2009; in revised form: 23 March 2009 / Accepted: 24 March 2009 /
`Published: 25 March 2009
`
`Abstract: Antisense molecules do not readily cross cell membranes. This has limited the
`use of antisense to systems where techniques have been worked out to introduce the
`molecules into cells, such as embryos and cell cultures. Uncharged antisense bearing a
`group of guanidinium moieties on either a linear peptide or dendrimer scaffold can enter
`cells by endocytosis and subsequently escape from endosomes into the cytosol/nuclear
`compartment of cells. These technologies allow systemic administration of antisense,
`making gene knockdowns and splice modification feasible in adult animals; this review
`presents examples of such animal studies. Techniques developed with PPMOs, which are
`an arginine-rich cell-penetrating peptide linked to a Morpholino oligo, can also be
`performed using commercially available Vivo-Morpholinos, which are eight guanidinium
`groups on a dendrimeric scaffold linked to a Morpholino oligo. Antisense-based techniques
`such as blocking translation, modifying pre-mRNA splicing, inhibiting miRNA maturation
`and inhibiting viral replication can be conveniently applied in adult animals by injecting
`PPMOs or Vivo-Morpholinos.
`
`Keywords: Antisense; Morpholino; PPMO; Vivo-Morpholino.
`
`1. Introduction
`
`Morpholino oligos are steric-blocking antisense molecules which bind to RNA and get in the way
`of cellular processes. These oligos have no electrical charge, do not interact strongly with proteins, and
`do not require the activity of RNase-H, Argonaute, or other catalytic proteins for their activity.
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`Molecules 2009, 14
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`Antisense molecules however do not readily cross cell membranes without delivery techniques, which
`has prevented their effective use in adult animals [1]. With delivery of Morpholino oligos achieved in
`vivo by Vivo-Morpholinos and Morpholino oligos linked to arginine-rich cell penetrating peptides
`(PPMOs), a longstanding barrier to applying Morpholino antisense techniques in adult animals has
`been overcome. Vivo-Morpholinos and PPMOs enter cells from the extracellular space and gain access
`to the cytosol and nuclear compartments. Antisense effects can be observed after systemic delivery of
`Vivo-Morpholinos or PPMOs.
`
`1.1. Uses of unmodified Morpholinos
`
`Effective techniques for delivering the oligos to the cytosol and nuclear compartments in tissue
`cultures have been developed, such as mechanical scraping [2], electroporation [3] or use of
`endosomal escape reagents [4]. Unmodified Morpholinos have been used routinely to block translation,
`modify splicing, inhibit miRNA activity and inhibit viral replication as well as more exotic RNA-
`blocking applications [5]. The Morpholino antisense structural type has been a revolutionary tool in
`developmental biology [6]. Success in embryos came by microinjecting Morpholino oligos into eggs
`or single or few celled zygotes, so that during cell division the Morpholinos were apportioned into
`daughter cells. This neatly avoided the problem of delivering the antisense separately into each cell of
`a many-celled organism [7]. In addition to their use to determine gene functions and interactions in
`embryos, Morpholino antisense oligos have been used for research into a broad range of diseases as
`well as several clinical trials (AVI BioPharma, Inc.). In most cases, carrying work from research to
`therapeutic applications of unmodified Morpholino oligos has been limited by difficulties with in vivo
`delivery [8]. However, delivery of relatively high doses of unmodified Morpholinos into dystrophic
`muscle in animal models of DMD has induced expression of functional Dystrophin in skeletal muscle
`[9,10] and unmodified Morpholinos are currently in clinical trial for treatment of DMD
`(http://clinicaltrials.gov/ct2/show/NCT00844597).
`
`1.2. Morpholino chemistry and nomenclature
`
`Morpholino oligos are manufactured from ribosides. The ribose ring is opened by oxidation, re-
`closed on ammonia and the product subsequently reduced to substituted morpholine. The base and the
`morpholine nitrogen are protected and the subunit is activated with a dimethylamino phosphoro-
`dichloridate. The activated subunits are added to a synthesis resin with washing, deprotection and
`activation steps for each activated base added. Oligos are cleaved from the resin and deprotected with
`ammonium hydroxide, then purified and quantitated, often followed by lyophilization and sterilization
`[11]. Substitutions such as peptides or the dendrimer scaffold for guanidinium may be added to the 3’
`morpholine nitrogen while the oligo is still attached to the synthesis resin. Alternatively, peptides may
`be added in the solution phase after resin cleavage and purification steps.
`The ends of a Morpholino oligo are described as 3’ and 5’, but these labels do not refer to properly
`numbered atoms of the Morpholino backbone. Instead the atom designations of natural nucleic acids
`are used by analogy to label Morpholino ends; nucleic acids have a 5’-methylene hydroxyl (often
`phosphorylated) at one end and a 3’-ring hydroxyl at the other end. The methylene attached to the
`morpholine ring is designated as the 5’ end of a Morpholino subunit and the morpholine nitrogen is
`designated as the 3’ end; this nomenclature is chosen in order to make designation of direction along
`
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`Molecules 2009, 14
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`the Morpholino oligo familiar to molecular biologists, who are accustomed to referring to the 5’ and 3’
`ends of RNA and DNA.
`
`1.3. Endocytosis and the barrier to entry into the cytosol/nuclear compartment
`
`Unmodified Morpholino oligos are endocytosed but have little to no activity against their RNA
`targets. Uptake by endocytosis does not mean the antisense oligos are reaching the cytoplasm or
`nucleus of the cell. Fluorescently-labeled Morpholinos can be detected as dim punctuate fluorescence
`within endosomes, but in most cells too little antisense escapes from the endosomes to the
`cytosol/nuclear compartment to be biologically active [2]. Morpholinos are not degraded in endosomes
`[12] but remain trapped. Many biological problems are best studied in adults and so a method that
`allows entry of antisense into most or all cells in an adult organism is very desirable. Ideally this
`systemic delivery could be accomplished by routine means such as intravenous or intraperitoneal
`injections. A technology that allows enough of the endocytosed oligos to escape from endosomes to
`have biological activity would enable in vivo delivery of oligos administered by systemic injection.
`Such technologies have now been developed: PPMOs and Vivo-Morpholinos. A PPMO may have an
`arginine-rich cell-penetrating peptide linked to either the 3’ end or the 5’ end of the Morpholino oligo
`[13]. The arginines have guanidinium moieties as part of their side chains, and the presence of these
`guanidiniums have been shown to increase cellular uptake of conjugated materials [14]. Studies of the
`mechanism of PPMO entry have shown that the cell-penetrating peptides offer two advantages over
`unmodified Morpholinos: enhanced uptake into endosomes and, critically, enhanced ensodomal escape
`[15]. A Vivo-Morpholino has an octaguanidinium dendrimer constructed on the 3’ end of a
`Morpholino oligo [16] (Figure 1).
`
`1.4. PPMOs
`
`The first effective chemically-mediated method for systemic delivery of Morpholino antisense was
`based on covalently linking the oligos to cell-penetrating peptides, using arginine-rich cell-penetrating
`peptides to deliver uncharged antisense molecules [17]. These molecules are called peptide-linked
`phosphorodiamidate Morpholino oligomers, or PPMO for short. Research to develop more safe and
`effective peptide sequences for PPMOs has been the focus of Hong Moulton’s group at AVI
`BioPharma Inc. [18,19], where preclinical work on PPMOs for Duchenne muscular dystrophy is
`currently ongoing. Studies on PPMOs have shown that uptake of the oligos is an energy-dependant and
`temperature-dependant process that can be prevented using endocytosis inhibitors; these characteristics
`indicate that uptake of the PPMOs is by endocytosis [20]. A fraction of endocytosed PPMOs escape
`from the endosome, entering the cytosol and nuclear compartment where they can block mRNA
`translation and modify pre-mRNA splicing [21]. Intravenous injection [22], intraperitoneal injection or
`intranasal administration [23] of PPMOs to mice have inhibited viral replication. Injections of PPMOs
`into transgenic mice carrying engineered splice-reporter genes have triggered expression of green
`fluorescent protein throughout the tissues [24].
`
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`Molecules 2009, 14
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`Figure 1. Structures of unmodified and delivery-enabled Morpholino oligos.
`Base1
`Base
`
`Basen
`
`3' end
`
`O
`
`NH
`
`N
`
`N
`OP
`O
`n-2
`
`Basen
`
`Gene Tools Morpholino
`
`5' end
`
`H2N
`O
`
`O
`
`N
`OP
`
`N
`O
`
`O
`
`N
`
`N
`OP
`O
`
`5' end
`
`O
`
`HO
`
`O
`
`O
`
`O
`
`N
`
`AVI BioPharma Morpholino (PMO)
`
`Base1
`
`O
`
`Base
`
`O
`
`N
`
`N
`OP
`O
`
`N
`
`N
`OP
`O
`
`3' end
`
`O
`
`NH
`
`NH
`NH2
`NH
`
`NH
`NH2
`NH
`NH2
`
`NH
`
`HN
`
`HN
`
`N
`
`N
`OP
`O
`n-2
`
`NH
`
`O
`
`N
`
`ON
`
`O
`
`N
`
`N
`
`Base1
`O
`
`N
`
`H2N
`O
`
`N
`
`Base
`
`O
`
`N
`
`Basen
`O
`
`N
`
`NH2
`NH2
`
`NH
`NH2
`NH
`
`HN
`
`HN
`
`HN
`
`NH2
`NH
`NH2
`NH
`
`O
`
`O
`
`N
`
`N
`
`N
`
`N
`N
`
`O
`O
`
`O
`
`N
`
`HN
`
`O N
`
`O
`
`N
`OP
`O
`
`N
`OP
`O
`
`N
`OP
`O
`n-2
`Gene Tools Vivo-Morpholino
`
`O
`
`HO
`
`O
`
`O
`
`O
`
`N
`
`Base1
`
`O
`
`Base
`
`O
`
`N
`
`N
`OP
`O
`
`N
`
`N
`OP
`O
`
`AVI BioPharma PPMO
`3'-conjugate with (RXRRBR)2XB- peptide
`
`Basen
`
`O
`
`N
`
`N
`
`N
`OP
`O
`n-2
`
`BXRBRRXRRBRRXR-NH-Ac
`
`Ac = acetyl,
`R = arginine,
`X = 6-aminohexanoic acid,
`B = beta-alanine
`
`AcHN-RXRRBRRXRRBRXB
`
`N
`
`AVI BioPharma PPMO
`5'-conjugate with (RXRRBR)2XB- peptide
`
`N
`
`Base1
`
`O
`
`N
`OP
`O
`
`N
`
`N
`OP
`O
`
`Base
`
`O
`
`Basen
`
`O
`
`NH
`
`N
`
`N
`OP
`O
`n-2
`
`Positively-charged arginine amino acid residues are not electrostatically attracted to uncharged
`antisense such as Morpholino oligos or peptide nucleic acid (PNA) oligos, so the positively charged
`arginines are free to interact with membranes [25]. At the end of an arginine amino acid’s side chain is
`a guanidinium group, which carries the amino acid’s positive charge. This charged guanidinium group
`is not only attracted to a phosphate because of the phosphate’s negative charge, it can also form two
`hydrogen bonds with the phosphate’s oxygens. This makes the attraction of a guanidinium and a
`
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`Molecules 2009, 14
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`phosphate an unusually strong non-covalent interaction [26]. It may be this strong interaction that
`distorts the endosomal membrane and renders it permeable.
`
`1.5. Vivo-Morpholinos
`
`Vivo-Morpholinos are Morpholino antisense oligos covalently linked to a molecular scaffold that
`carries a guanidinium group at each of its eight tips. These custom-sequence antisense molecules
`enable Morpholino applications in adult animals. Vivo-Morpholinos have been shown effective in
`mice [27,28] and are being tested in rats, adult zebrafish and various organ explants.
`To make a Vivo-Morpholino, the scaffold that will hold the guanidinium groups is added to the
`morpholine nitrogen at the 3’-end of a Morpholino oligo while the oligo is still bound at its 5’ end to
`the synthesis resin. Subsequent treatment with ammonia cleaves the Vivo-Morpholino from the
`synthesis resin, de-protects the Morpholino’s bases and de-protects the eight tips of the 3’-terminal
`scaffold. Next, treatment with O-methyl isourea converts the eight terminal amino groups to
`guanidinium groups [16].
`
`2. Targets and outcomes of Morpholino experiments
`
`Morpholino oligos bind to complementary RNA. If a molecular process normally occurs at the site
`where the Morpholino is bound to RNA, the Morpholino might be suitably positioned to get in the way
`of that process and prevent it from occurring. This mechanism is called steric blocking [29]. The effect
`of a Morpholino on a cell depends on where the Morpholino binds to the RNA, which determines
`whether it can block a process and which process it blocks. There are three common applications for
`Morpholino oligos: blocking translation of mRNA, modifying splicing of pre-mRNA or inhibiting
`miRNA activity [5]. In addition, work has been done on viral targets such as internal ribosomal entry
`sites [3] and cyclization sequences [30]; much of the reported work with PPMOs has involved viral
`targets [31]. Exotic targets have also been explored, such as binding to slippery sites to trigger
`ribosomal frameshifting [32] or binding to ribozymes to prevent catalytic cleavage of RNA [33].
`
`2.1. Translation blocking
`Translation blocking is the simplest method for knocking down gene expression with a Morpholino.
`When making a protein by cap-dependant translation, the small subunit of a ribosome binds to the 5’
`cap of an mRNA and, along with some other proteins, forms an initiation complex. The initiation
`complex moves along the 5’ untranslated region (UTR) of the mRNA until a start codon is reached. At
`the start codon, the large subunit of the ribosome docks to small ribosomal subunit and then the
`process begins of linking together amino acids to form the new protein. Morpholinos that block the
`journey of the ribosomal initiation complex from the 5’-cap to the start codon, halting translation of
`the mRNA before the linking of amino acids can start. These Morpholinos can bind to RNA targets
`anywhere in the 5’-UTR through the start of the coding region, so long as the oligo binds onto the start
`codon or upstream (to the 5’ direction) of the start codon [34].
`The activity of a translation blocking oligo is assayed by an immunochemical method, typically
`Western blotting. Halting translation of a protein will not immediately cause a detectable change in the
`protein’s signal on a Western blot. Some preexisting protein must be degraded over time before the
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`protein signal decreases. The time needed between knockdown and assay varies with the protein, as
`different proteins have different stabilities in cells.
`
`2.2. Splice modification
`
`After a pre-mRNA is transcribed from a DNA template, it goes through processing in the nucleus to
`form a mature mRNA. This processing typically includes adding a 5’ cap, splicing out introns and
`ligating together exons, and adding a 3’ poly-A tail. Splice modification involves binding Morpholinos
`to targets on a pre-mRNA (an unspliced mRNA) to redirect the spliceosome to a new pattern of
`splicing and produce a mature mRNA sequence that differs from the sequence produced in the absence
`of the Morpholino. Usually Morpholinos are bound to mostly-intronic targets at the boundaries
`between exons and introns. These targets prevent the binding of molecules that direct the spliceosome
`to splice sites. Typical results of splice modification include removal of a targeted exon or inclusion of
`the first or last intron in the mature mRNA [35].
`Another class of targets consists of the binding sites on RNA where splice-regulation proteins
`normally bind, such as exonic splice enhancers or intronic splice suppressors. These proteins are
`important in directing alternative splicing, a natural process by which cells make alternative forms of
`mRNA from a single gene. For some targets, preventing a splice-regulatory protein from binding is an
`efficient method by which Morpholino can change the pattern of RNA splicing and therefore change
`the sequence of a mature mRNA [10,36].
`Splice modification might produce an mRNA that is expressed as an altered protein and that protein
`might retain some activity and might still bind antibodies that would also bind to the unaltered protein.
`Because of this, immunochemical techniques used to detect knockdowns by translation blocking
`Morpholinos often do not work when used for detecting activity of splice-modifying Morpholinos. The
`activity of splice-modifying oligos is typically assayed by RT-PCR from primers that bind on either
`side of the altered sequence. If the altered splice leaves downstream sequence in-frame, the RT-PCR
`product will appear at a different location on an electrophoretic gel. Activation of a cryptic splice site
`can result in an RT-PCR fragment of unexpected size [37]. If the altered splice shifts the downstream
`reading frame, appearance of a premature termination codon often results in nonsense-mediated decay
`of the modified RNA, so the wild-spliced RT-PCR product band might dim on the electrophoretic gel
`without the corresponding splice-modified RT-PCR product appearing as a new band in a different
`position.
`
`2.3. Inhibiting microRNA
`
`A mature micro-RNA (miRNA) is a short strand of RNA bound to a protein complex which
`changes the expression of other genes by mechanisms that include cleaving or inhibiting translation of
`the target RNAs. We will discuss miRNAs of animals; there are some differences in plants. The target
`RNA must have sites at least partially complementary to the miRNA. Usually (but not always [38]) the
`target sites for miRNAs are in the 3’-UTR of mRNAs. miRNA is formed when a short stem-loop
`(hairpin) with the appropriate geometry forms in a newly-transcribed RNA. In the nucleus, the hairpin
`is recognized and cleaved by the Drosha nuclease, which cleaves both strands of the stem and leaves a
`few single-stranded overhanging bases. The resulting stem-loop is exported to the cytosol where it is
`recognized and cleaved by the Dicer nuclease, which removes the loop and also leaves a few single-
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`Molecules 2009, 14
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`stranded overhanging bases. The resulting short double-stranded RNA is loaded onto a protein
`complex called the RNA-induced silencing complex (RISC), where a protein in the Argonaute family
`binds to one strand, called the guide strand, and cleaves the other strand. The cleaved strand
`dissociates and diffuses away, leaving an active miRNA-RISC complex [39].
`Morpholinos can bind to the miRNA guide strand on RISC, preventing the guide strand from
`recognizing its targets on mRNAs [40]. Morpholinos can bind to the immature RNA hairpin,
`preventing Drosha from releasing the stem-loop from the rest of the transcript. Morpholinos can bind
`to the stem-loop, preventing Dicer from cleaving the loop from the stem [41]. A typical strategy is to
`design a 25-base Morpholino to bind the entire guide strand sequence (around 21 bases) and slightly
`into the loop sequence, allowing both inhibition of mature miRNA activity on RISC and inhibition of
`miRNA maturation by blocking Dicer. Another option is protecting a mRNA target. By binding to its
`complementary miRNA, a Morpholino blocks RISC from accessing the mRNA, thus relieving
`translation of the mRNA from inhibition by the miRNA and protecting the mRNA from cleavage by
`miRNA-directed Argonaute activity [42]. An advantage of target protection is that only the expression
`of the target gene is altered, while inhibiting maturation and activity of a miRNA can alter the
`expression of many mRNAs.
`
`3. PPMOs
`
`PPMOs are covalent conjugates of Morpholino oligos with cell-penetrating peptides. The peptide
`may be attached at the 3’ or 5’ end of the Morpholino oligo. Successful cell-penetrating peptides have
`contained arginine residues. The peptides may be composed of the alpha amino acids common in
`natural proteins or may contain other amino acids such as (cid:69)-alanine or 6-aminohexanoic acid. Some
`cell-penetrating peptide sequences discussed in this section include (RXR)4B-, (RXR)4XB-,
`(RXRRBR)2XB- and (RX)8B-, with the dash at the peptide carboxy terminus representing the link to
`the Morpholino oligo. For these structures, R = arginine (L-arginine unless otherwise noted ), B = (cid:69)-
`alanine, and X = 6-aminohexanoic acid. Chiral amino acids may be in D or L forms, with the D forms
`chosen to resist proteolytic degradation, as in the two terminal arginines of the peptide
`(DR)2R2QR2K2RF2C-. A procedure for solution-phase conjugation of Morpholino antisense oligos to
`arginine-rich peptides has been published [21].
`The literature of PPMO therapeutic applications has been reviewed recently [8]. This section will
`focus on PPMO papers published since that review was prepared, especially seeking efficacy
`comparisons between unmodified Morpholinos and PPMOs and assessments of toxicity or
`immunogenicity of the PPMOs. Where many sequences of cell-penetrating peptides were reported in a
`paper, we will discuss the most effective of the peptides; for more detailed structure-activity
`comparisons, refer to the primary papers.
`
`3.1. Screening cell-penetrating peptides in EGFP mice
`
`A set of 14 cell-penetrating peptides were attached to the 5’ ends of Morpholinos targeting a mutant
`splice site in human (cid:69)-globin (IVS2-654) and administered to EGFP-654 mice, which carry a
`transgene with EGFP interrupted by an aberrantly-spliced human (cid:69)-globin mutant intron (IVS2-654).
`Administration of different peptides resulted in different biodistributions. Because the cell-penetrating
`peptide (RXRRBR)2XB- was effective at correcting IVS2-654 splicing in heart, diaphragm and
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`quadriceps, important target muscles for Duchenne muscular dystrophy (DMD) treatment, and did not
`cause toxicity at 12 mg/kg daily for four days, it was selected for intensive study. This PPMO also
`modified splicing in smooth muscles of the gut [24].
`To determine persistence of effects, 12 mg/kg daily for four days of the (RXRRBR)2XB- PPMO
`targeting IVS2-654 was administered to a group of EGFP-654 mice and mice were sacrificed and
`assessed periodically for 12 weeks. As shown by RT-PCR of cardiac muscle one day after the final
`injection, 90% of transcripts were splice-corrected. This decreased to 10% splice correction at six
`weeks. In diaphragm, splice correction decreased from 100% at 2-3 weeks after treatment to 10%-40%
`at 12 weeks. In quadriceps, splicing was still 100% corrected at 12 weeks. [24]
`
`3.2. Duchenne muscular dystrophy
`
`Duchenne muscular dystrophy (DMD) arises from some mutations of the human dystrophin gene.
`A potential treatment for this disease is the use of steric-blocking oligos to redirect splicing, skipping
`exons to remove early stop codons or to correct the reading frame disrupted by frameshift mutations.
`Several mouse models of DMD have been developed, the first and most popular being the mdx mouse,
`which has a premature stop codon in exon 23. Unmodified Morpholino antisense is currently in
`clinical trial for DMD. However, unmodified Morpholinos do not enter the heart at effective
`concentration and affect dystrophin splicing, even after repeated doses as high as 100 mg/kg in mdx
`mice [9]. For the three papers reviewed below, measurements of mRNA exon 23 skipping and
`Dystrophin concentrations after systemic delivery of PPMOs in mdx mice are summarized in Table 1.
`
`3.2.1. Studies with the (RXRRBR)2XB- cell-penetrating peptide PPMOs
`
`A PPMO with the (RXRRBR)2XB- peptide conjugated to the 3’ end of a Morpholino sequence
`designed to cause splice-mediated excision of dystrophin exon 23 was injected iv into mdx mice at 12
`mg/kg daily for four days. RT-PCR of RNA from heart tissue found that 70% of the dystrophin mRNA
`lacked exon 23 one day after the last treatment, decreasing to 50% at two weeks and 20% at seven
`weeks. At nine weeks after treatment, exon 23-skipped mRNA was still detected. In diaphragm and
`quadriceps, almost complete skipping of exon 23 persisted through nine weeks [24].
`In-gel immunostaining revealed that in heart 30% of normal dystrophin levels were reached at 2-3
`weeks and 15% remained at seven weeks after treatment. In diaphragm and quadriceps 40%-50% of
`normal dystrophin remained up to 17 weeks after treatment [24].
`A PPMO with the (RXRRBR)2XB- peptide on a Morpholino targeting dystrophin exon 23 in the
`mdx mouse was compared with an unmodified Morpholino of the same sequence. Either the
`Morpholino or the PPMO were injected im at 30 mg/kg into the tibialis anterior (TA) of adult mdx
`mice. The PPMO induced strong dystrophin expression in 85% of the fibers of the TA while the
`Morpholino induced dystrophin expression in 14% of TA fibers [43].
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`Table 1. Comparison of systemic delivery of PPMOs for DMD.
`
`PPMO with (RXRRBR)2XB- peptide on 3’ end [24], 12 mg/kg iv tail vein, daily for 4 d
`Dystrophin mRNA with exon 23 skipped
`Dystrophin protein concentration
`Heart:
`Heart:
`70%, 1d post-treat;
`30% dystrophin 2-3wk post-treat;
`50%, 2wk post-treat;
`15% dystrophin 7wk post-treat.
`Diaphragm & quadriceps:
`20%, 7 wk post-treat.
`Diaphragm & quadriceps:
`40%-50% dystrophin, 17 weeks post-treat.
`~100% thru 9 wk post-treat.
`PPMO with (RXRRBR)2XB- peptide on 5’ end [43], 30 mg/kg iv retro-orbital, single dose
`Dystrophin mRNA with exon 23 skipped
`Dystrophin protein concentration
`Heart:
`Heart:
`63%, 2wk post-treat.
`58% dystrophin, 2wk post-treat.
`Skeletal muscle:
`Skeletal muscle:
`80%-86%, 2wk post-treat.
`91%-100% dystrophin, 2wk post-treat.
`PPMO with (RXRRBR)2XB- peptide on 5’ end [43], 30 mg/kg iv retro-orbital, once every two weeks for
`three months (six times)
`Dystrophin protein concentration
`Dystrophin mRNA with exon 23 skipped
`Heart:
`Heart:
`~100% 2wk post-treat.
`72%, 2wk post-treat.
`Skeletal muscle:
`Skeletal muscle:
`~100% 2wk post-treat.
`85%-92%, 2wk post-treat.
`PPMO with (RXR)4XB- peptide [44], 25 mg/kg iv tail vein, single dose
`Dystrophin mRNA with exon 23 skipped
`Dystrophin protein concentration
`Heart:
`Heart:
`50%, 3wk post-treat.
`10%-20% dystrophin, 3wk post-treat.
`Skeletal muscle:
`Skeletal muscle:
`Near 100%, 3wk post-treat.
`25%-100% dystrophin, 3wk post-treat.
`Percent protein and mRNA are compared with wild-type muscle as 100%.
`
`Single retro-orbital iv injections of the 30 mg/kg PPMO were administered to mdx mice and
`dystrophin expression was assessed two weeks later. Similar injections of the unmodified Morpholino
`induced dystrophin expression in 5% of skeletal muscle fibers. The PPMO induced strong dystrophin
`expression in 100% of skeletal muscle fibers and near-normal dystrophin levels were found by
`Western blot. RT-PCR of PPMO-treated mice showed 80%-86% of dystrophin transcripts in skeletal
`muscle were missing exon 23, demonstrating PPMO activity. Unmodified Morpholino induced no
`detectable dystrophin expression in cardiac muscle fibers. The PPMO induced dystrophin expression
`in 94% of cardiac muscle fibers and 58% of normal dystrophin levels was found by Western blot. RT-
`PCR revealed that 63% of dystrophin transcripts in cardiac muscle were missing exon 23 [43].
`To assess longer-term treatment, mdx mice were iv injected retro-orbitally with PPMO at 30 mg/kg
`once every two weeks for three months, totaling six injections per mouse. Two weeks after the last
`injection, dystrophin was found in 100% of muscle fibers including smooth muscle of the small
`intestine. Dystrophin levels resembled those of normal mouse tissue. In cardiac muscle exon skipping
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`was improved by this dosing regimen, with 72% of dystrophin mRNA lacking exon 23 and strong
`dystrophin protein expression comparable to normal heart [43].
`No damage to kidney or liver was detected by histological exams after PPMO treatment. Alkaline
`phosphatase and creatinine were not altered by PPMO treatment. Inflammatory cells did not
`accumulate in muscles and no antibodies reactive with PPMO were found in serum, indicating that the
`PPMO was not immunogenic [43].
`
`3.2.2. Study with the (RXR)4XB- cell-penetrating peptide PPMO
`
`Single tail vein injections of 25 mg/kg PPMO with peptide sequence (RXR)4XB- and a Morpholino
`sequence designed to cause excision of exon 23 were administered to mdx mice. Three weeks later,
`skeletal muscles were immunostained and showed near normal levels of dystrophin in most muscles
`analyzed. Western blot analysis found dystrophin concentrations ranging between 25% to 100% of
`normal in skeletal muscles. RT-PCR revealed almost total skipping of exon 23 in all skeletal muscles
`analyzed [44].
`In heart muscle RT-PCR showed that about 50% of dystrophin transcripts lost exon 23.
`Immunostaining revealed dystrophin-positive fibers were widely distributed. By Western blotting,
`dystrophin concentrations between 10% and 20% of normal levels in heart were found [44].
`PPMOs with the cell-penetrating peptides (RXR)4XB- and (RXRRBR)2XB- were injected iv and
`their efficacy compared. While both delivered antisense activity to skeletal and cardiac muscle, the
`(RXR)4XB- did so more effectively [44].
`To detect toxicity, analysis of the histology of liver and kidney and assays of some serum
`components were performed on samples from mdx mice that had been treated with 25 mg/kg PPMO.
`No overt signs of tissue damage in kidney or liver were revealed in haematoxylin and eosin stained
`tissues and there was no change in the number of infiltrating cells compared with untreated mdx mice.
`In the PPMO treated mice, levels of aspartate aminotransferase (AST) and alanine aminotransferase
`(ALT) went from levels typical of mdx mice to levels typical of normal control mice. Serum levels of
`urea and creatinine did not change with PPMO treatment [44].
`Weekly 6 mg/kg tail vein injections of the same PPMO for three weeks resulted in less exon
`skipping than the single 25 mg/kg tail vein injections, with the clearest decreases in efficacy in
`diaphragm, abdominal wall muscles and heart [44].
`
`3.3. (cid:69)-thalassemia
`
`(cid:69)-thalassemia is a common inherited genetic disease of humans. The ability of PPMOs to redirect
`splicing in a specific splice mutant of human (cid:69)-globin was assessed in heterozygous knock-in mice, in
`which two adult murine (cid:69)-globin genes ((cid:69)-major and (cid:69)-minor) were deactivated. This study used the
`cell-penetrating peptide (DR)2R2QR2K2RF2C- covalently linked to a Morpholino targeting the aberrant
`5’ splice site created by the mutation IVS2-654 (C>T). Blocking this splice site restores normal
`splicing of (cid:69)-globin transcripts bearing this IVS2-654 (C>T) mutation. Mice were treated with 25
`mg/kg injections with daily injections for four days followed by three rest days, then this cycle was
`repeated. The experiment terminated on day 19 [45].
`No significant liver or kidney toxicity were detected in PPMO-treated mice compared to saline-
`treated mice by standard biochemical tests (AST, ALT, BUN, CREA), nor was there significant weight
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`loss. Inflammatory response was assessed by RT-qPCR of IFN-(cid:74) and LI-12a cytokines; there was no
`significant change detected in their expression comparing saline-injected with PPMO-injected mice.
`Treatment of mice with a boost of PPMO a month after they were initially primed with 12 to 16 doses
`of the PPMO did not result in an IL-12 response as assessed by immunoassay for IL-12 on sera from
`days 3, 7 and 28 after boosting. Remaining sera from days 3, 7 and 28 after boosting was tested by
`ELISA for antibodies to the cell-penetrating peptide; no such antibody was detected. Lymphocytes
`from mice that had been treated 10 months earlier with either saline or PPMO were cultured ex vivo,
`challenged with PPMO and tested for induction of IFN-(cid:74) or antibody to the cell-penetrating peptide;
`neither response was detected [45].
`The level of (cid:69)-globin restoration theoretically achievable under these experimental conditions was
`fairly low. First, the mice were heterozygous for the human knock-in, so the human hemoglobin could
`only be restored to 50% normal concentration from the human gene. The duration of the experiment
`was 19 days but murine erythrocytes normally reside in blood for 60 days, so only a maximum of 1/3
`of the erythrocytes could be replaced by the end of the experiment. Combining this with the 50%
`expression of human (cid:69)-globin from the heterozygotes, the fraction of hemoglobin containing human (cid:69)-
`globin theoretically achievable by the end of the experiment drops just under 17%. The level of
`chimeric mouse-human hemoglobin reached about 1-5% of the total hemoglobin in treated mice by
`day 19 [45].
`
`3.4. Antiviral applications
`
`Typically the highest antiviral efficacies are achieved with pre-infection administration