Peptides 67 (2015) 1–11

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Peptides journal homepage: www.elsevier.com/locate/peptides

Review

Peptidic tools applied to redirect alternative splicing events Martínez-Montiel Nancy a , Rosas-Murrieta Nora b , Martínez-Contreras Rebeca a,∗ a Laboratorio de Ecología Molecular Microbiana, Centro de Investigaciones en Ciencias Microbiológicas, Instituto de Ciencias, Benemérita Universidad Autónoma de Puebla, Mexico b Laboratorio de Bioquímica y Biología Molecular, Instituto de Ciencias, Benemérita Universidad Autónoma de Puebla, Mexico

a r t i c l e

i n f o

Article history: Received 14 October 2014 Received in revised form 5 February 2015 Accepted 26 February 2015 Available online 6 March 2015 Keywords: Splicing mRNA Mutations Cell-penetrating peptide Peptide nucleic acid

a b s t r a c t Peptides are versatile and attractive biomolecules that can be applied to modulate genetic mechanisms like alternative splicing. In this process, a single transcript yields different mature RNAs leading to the production of protein isoforms with diverse or even antagonistic functions. During splicing events, errors can be caused either by mutations present in the genome or by defects or imbalances in regulatory protein factors. In any case, defects in alternative splicing have been related to several genetic diseases including muscular dystrophy, Alzheimer’s disease and cancer from almost every origin. One of the most effective approaches to redirect alternative splicing events has been to attach cell-penetrating peptides to oligonucleotides that can modulate a single splicing event and restore correct gene expression. Here, we summarize how natural existing and bioengineered peptides have been applied over the last few years to regulate alternative splicing and genetic expression. Under different genetic and cellular backgrounds, peptides have been shown to function as potent vehicles for splice correction, and their therapeutic benefits have reached clinical trials and patenting stages, emphasizing the use of regulatory peptides as an exciting therapeutic tool for the treatment of different genetic diseases. © 2015 Elsevier Inc. All rights reserved.

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alternative splicing mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Small molecules designed to specifically modulate splicing events. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Anomalous splicing events corrrected using ASOs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Peptidic carriers designed to introduce nucleic acids into eukaryotic cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CPP carriers as a powerful tool for splicing redirection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Splicing arrest using peptide inhibitors of protein kinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Natural protective peptides that modulate alternative splicing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Peptides blocking protein–protein interaction stall the spliceosome. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2 2 2 2 4 6 8 8 8 9 9 9

Abbreviations: ANP, atrial natriuretic peptide; ASBNP, alternatively spliced BNP; ASO, antisense oligonucleotides; BNP, B-type natriuretic peptide; CBD, calmodulin binding domain; CPP, cell penetrating peptides; DMD, Duchenne muscular dystrophy; ESE, exonic splicing enhancer; ESS, exonic splicing silencer; GFR, glomerular filtration rate; IAS, intronic activators of splicing; ISE, intronic splicing enhancer; ISS, intronic splicing silencer; LMN, lamin; NA, nucleic acids; NLS, nuclear localization signal; ON, oligonucleotides; Pen, penetratin; PID, primary immunodeficiency disease; PMO, phosphorodiamidate morpholino oligos; PNA, peptide nucleic acid; PPMO, peptidePMO; PTD, protein transduction domains; siRNA, small interfering RNA; snRNA, small nuclear ribonucleic acid; snRNP, small nuclear ribonucloprotein particles; SCO, Splice-oligonucleotides; SR, serine–arginine rich domains; SSO, splice-switching oligonucleotides; TP, transportan. ∗ Corresponding author at: Edificio 103 J, Ciudad Universitaria, Col. San Manuel, Puebla, Pue. CP 72570, Mexico. Tel.: +52 222 2295500x2529. E-mail address: [email protected] (M.-C. Rebeca). http://dx.doi.org/10.1016/j.peptides.2015.02.006 0196-9781/© 2015 Elsevier Inc. All rights reserved.

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Introduction Defects in splicing play a significant role in several diseases and genetic disorders [73,106]. Due to the importance of this mechanism on regulating gene expression, splicing is consequently an emerging therapeutic target. One of the first attempts to regulate splicing was the development of synthetic oligonucleotides (ON) designed to target specific genes in order to regulate alternative splicing events [1,18]. The main limitation of the use of many types of synthetic ON and their analogs as therapeutic antisense agents has been their poor cellular delivery. In order to achieve successful ON transfection, efficient delivery vectors are generally necessary. Many types of such vectors have been designed to aid ON delivery both for cell culture and in vivo [41,70]. Amongst such strategies, conjugation to cell penetrating peptides (CPP) has received attention recently [58,78]. At present, multiple drugs that rely on the antisense splicing redirection principle involving the use of CPPs as carriers are in advanced phases of clinical trials [80,108,110]. Characteristically, CPPs are short, cationic peptides that often have amphipathic properties and share structural features with antimicrobial peptides [47]. These CPPs, developed as vectors for cargo delivery into eukaryotic cells, have been extensively applied as delivery agents for splicing regulatory molecules [11,31]. According to their nature, some CPPs exhibit antimicrobial action toward bacteria, parasites, and fungi in vitro besides the effect on splicing regulation. Such antimicrobial activity is observed at higher concentrations than the one required for translocation across eukaryotic membranes [46]. In this review, we will discuss not only the evidence related to CPPs, but we will also present other efforts that target splicing regulation by the use of natural and synthetic molecules, inspired in nature-existing peptides. Alternative splicing mechanism Pre-mRNA splicing is a nuclear process that occurs in all eukaryotes whereby the intervening sequences in nascent transcripts, called introns, are removed while the flanking coding sequences are joined together to produce a mature mRNA molecule that is subsequently exported to the cytoplasm where it serves as a template used in protein translation. The splicing mechanism is catalysed by a large ribonucleoprotein complex called spliceosome [102]. The core components of the spliceosome include four ribonucleoprotein particles (snRNP): U1, U2, U4/U6 and U5. Each particle contains the correspondent RNA molecule (snRNA) and a set of specific and common proteins [111]. The spliceosome complex also contains multiple auxiliary factors that are essential for complex assembly and splicing catalysis [54]. These regulatory factors include the SR family of proteins, which are commonly known as splicing facilitators [64] and the heterogeneous group of hnRNP proteins [72]. The assembly of the splicing factors on the pre-mRNA conforms the active spliceosome that would be ready for catalysis [74]. RNA splicing depends on the proper recognition of exons, which is a challenge, given that the average size for a human exon is 100–300 nucleotides. Exon recognition is guided in part by conserved sequence elements at exon–intron boundaries, named 5 and 3 splice sites, but it also depends on sequence elements within exons and on intronic elements distinct from the splice sites, which have been divided into four functional categories: exonic splicing enhancers (ESEs), exonic splicing silencers (ESSs), intronic splicing enhancers (ISEs) also known as intronic activators of splicing (IASs) and intronic splicing silencers (ISSs). These elements and the factors that recognize them are employed for the definition of introns and exons (Fig. 1A). This combinatorial control regulates splicing decisions [40]. Alternative splicing expands the coding capacity of the human genome given the ability to generate diverse protein isoforms from a single gene. In many cases, the specific function of

each isoform is not yet understood. There are several types of alternative splicing (Fig. 1B). Inclusion or skipping of a complete exon is the most common case of alternative splicing. In some cases, a whole intron is removed or retained to generate two different isoforms. A portion of an exon can also be present or absent in the final isoform. Additionally, different transcription start sites or polyadenylation signals could be used according to the spliced sequences. A combination of more than one alternative splicing event can occur in the same gene [53]. According to the information obtained from the Human Genome Project, approximately 75% of the human genes undergo some type of splicing [61]. Data from the Human Gene Mutation Database suggest that near 10% of the mutations annotated impinge on splice sites [103]. However, these percentage underestimates the number of mutations that affect splicing given that they do not consider the intronic and exonic regulatory elements described before. In addition, mutations associated with disruption of recognition sites for regulatory factors can result in global splicing defects, which can generate very different phenotypes. With all these considerations, it is reasonable to suggest that a large fraction of all human mutations affect splicing. Therapeutic tools have been developed to correct some of these anomalous splicing events. Some examples of the developments involving peptidic molecules are presented here. Small molecules designed to specifically modulate splicing events. The development of strategies to study the mechanisms that regulate gene expression has focused on modifying or modulating this process at the post-transcriptional level using nucleic acids (NA) or nucleic acids analogs such as short oligonucleotides (ONs), designed either to silence or to enhance gene expression. Nucleic acids analogs could be single stranded antisense oligonucleotides (ASOs) designed as a complementary molecule that targets a specific mRNA blocking gene expression both in vitro and in vivo, through the inhibition of translation or by enzymatic cleavage of the target mRNA. Moreover, there are double stranded RNAs such as short or small interfering RNAs (siRNAs) used to hydrolyze mRNA and to avoid the translation of a specific messenger into protein. These oligonucleotides could be modified to improve their properties as modulators of gene expression conffering an increased resistance to nucleases [29], the most common modifications are summarized in Fig. 2A [24]. Interestingly, some of these have been successfully tested on the splicing process. A modified NA analog is a non-RNase-H inducing 2 -O-methyl oligonucleotide (2 -OMe), useful to conserve the target RNA intact in splicing redirection [51]. PNA (peptide nucleic acid) molecules, which are neutral oligonucleotide analogs, are considered potential antisense drugs, and could be excellent candidates for gene therapy in AIDS patients [85] or in redirecting splicing [25]. Morpholino oligomers or phosphorodiamidate morpholino oligos (PMOs) are antisense nucleic acid analogs frequently used to modulate pre-mRNA splicing by originating a steric antisense blockage without activating RNaseH. PMOs have proven to be effective agents inside cells [110] and have been effective inducing exon-skipping in Duchenne muscular dystrophy (DMD) patients [20]. Some of the applications of ASOs are further discussed. Anomalous splicing events corrrected using ASOs It has been mentioned that pre-mRNA splicing is a tightly coordinated process that can be disrupted by ONs in a highly specific manner to suppress aberrant splicing, redirect exon recognition or intron removal and it can also bypass nonsense or frame-shifting mutations to alter isoform ratios [1]. ASOs can bind to target sequences in mRNA by standard nucleic acid base-paring. In other cases, AMOs can bind to a target site and blocking the interaction

M.-M. Nancy et al. / Peptides 67 (2015) 1–11

3

Fig. 1. Alternative splicing mechanism and events. (A) Splicing factors recognize different sequences along the pre-mRNA. Conserved recognition sequences include intron boundaries named 5 ss and 3 ss. These elements interact with the spliceosome, recruiting snRNPs (U1, U2, U4, U5 and U6). Other regulatory elements are embedded in the exon (ESE, ESS) or in the intron (ISE) and could be recognized by proteins of the SR and/or hnRNP families. (B) Possible alternative splicing events. Coding sequences are shown as colored thick lines, non-coding sequences represented as black thin lines. The name for each type of event is written in the left.

of spliceosomal components with the RNA. In this case, ASOs target selected splicing sites or regulatory elements in pre-mRNAs to reprogram the splicing process. Aberrant pre-mRNA splicing is involved in various diseases and it has been described that the use

of anomalous splice sites can be corrected using ON with potential therapeutic applications [13]. For example, ASO-mediated splicing modulation approaches are a viable strategy for the treatment of primary immunodeficiency diseases (PIDs), a heterogeneous

Fig. 2. Oligonucleotide analogs delivered by cell-penetrating peptides (CPPs). (A) Oligonucleotide analogs: phosphorothioate (PS) linkages, locked nucleic acid (LNA), 2 O-methyl oligonucleotide (2 -OMe), phosphorodiamidate morpholino oligonucleotide (PMO), peptide nucleic acid (PNA) [24,51]. (B) Conjugation of CPP-oligonucleotide analog. (1) Conjugation of PNA (NH2 -Cys(NPys)-Lys-CCTCTTACCTCAGTTACA-Lys-amide) to a stearyl-(RxR)4 peptide by disulfide linkage [58,95]. (2) Tat-PNA conjugate [5]. (3) Modified conjugate Pen-s-s-PNA [5].

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group of genetic disorders that result from mutations in genes involved in development and maintenance of the immune system. The origin of several PIDs corresponds to splicing mutations and nonsense mutations that can be manipulated by ASOs (reviewed in [28]). Moreover, strategies involving ASOs have been successfully tested in the treatment of ␤-thalassemia (␤-globin gene), DMD (dystrophin gene), spinal muscular atrophy (SMN2 gene), cystic fibrosis, block HIV replication and certain types of cancer by targeting Bcl-X[15,26,37,48,56]. The molecular insights for some of these applications are further presented. ␤-Thalassemia is a genetic disorder that can be caused by over 200 mutations in the ␤-globin gene, causing a deficiency of ␤-globin chains, leading to the reduction or the absence of adult hemoglobin A [65]. Some of the most frequent mutations cause aberrant splicing of intron 1 (IVS1-110, IVS1-6 and IVS1-5) or intron 2 (IVS2-1, IVS2-654, IVS2-705 and IVS2-745) of the human ␤-globin gene. Such mutations create an aberrant 5 splice site and activate a common cryptic 3 splice site at position 579 in intron 2 promoting the production of aberrantly spliced mRNA, aberrant translation and ␤thalassemia. Hybridization of a steric-block ON masks the mutated site, reorients the splicing machinery toward the complete removal of the intron, and leads to the production of a functional, fully spliced mRNA [13,87]. Stimulation of exon inclusion by antisense oligonucleotides is an alternative mechanism being examined also for the potential treatment of spinal muscular atrophy [40]. Another target for ASOs has been Bcl-x, which is a member of the bcl-2 family of apoptosis regulators. Alternative splicing of the bclx pre-mRNA generates two proteins with opposite functions from two main transcripts, a long (Bcl-xL) and a short (Bcl-xS) isoforms, encoding proteins with antagonistic functions. Bcl-xL inhibits apoptosis through heterodimerization with pro-apoptotic proteins whereas Bcl-xS shows pro-apoptotic properties. Using chimeric compounds PNA-SR that included Ser-Arg (SR)-rich domains of SR proteins can efficiently modulate splice site selection in the bclx gene in vivo and induce apoptosis in HeLa cells [112]. On the other hand, the up-regulation of bcl-x gene may be a common mechanism by which several types of cancer increase survival. Targeting bcl-x SSO (splice-switching oligonucleotides) using a lipid nanoparticle redirected bcl-x splicing and reduced tumor burden in melanoma lung metastases. The use of the upstream 5 alternative splice site of bcl-x exon 2 produces Bcl-xS, which directly inhibits the pro-apoptotic Bcl-xL and Bcl-2 proteins. Targeting the downstream (bcl-xL) splice site with a 2 -O-Me redirects the splicing machinery to the upstream (bcl-xS) splice site, converting it into a pro-apoptotic molecule to reduce tumor growth [14]. Considering that up to 50% of human disease-causing mutations affect splicing, the SSO approach is emerging as a promising alternative to gene therapy. Important efforts have been made in the delivery of oligonucleotides to the brain for the cure of neurodegenerative disorders, including diseases like ataxia telangiectasia, fronto-temporal dementia and Parkinsonism linked to chromosome 17 or neurofibromatosis (reviewed in [99]) together with the experience gained in the successful design of exon-skipping AONs for the cure of DMD will hopefully make personalized exon skipping therapies for several neurogenetic disorders a reality in the next few years. Peptidic carriers designed to introduce nucleic acids into eukaryotic cells The potential for therapeutic application of ASOs or SSOs to modulate pre-mRNA splicing is increasingly evident in a number of diseases. However, the primary drawback of this approach is poor cell and in vivo oligonucleotide uptake efficacy. Biological activities can be significantly enhanced through the use of synthetically conjugated CPPs [98].

To achieve the modulatory effect on gene expression, any type of nucleic acid or NA analog must enter cells or tissues through the hydrophobic plasma membrane [66]. NA share inadequate bioavailability to enter cells due to their negative charge, large molecular weight and low hydrophobicity that prevent these molecules to cross cellular membranes, unless they associate with a suitable delivery vehicle [62]. Several strategies to introduce the NA or analogous into the cell have been developed such as microinjection, electroporation, liposomes and viral vectors [22]. Any of these have some drawbacks as low efficiency, high toxicity, poor bioavailability and specificity. Therefore, alternative ON delivery systems have been described such as CPPs, which are a group of efficient non-viral delivery vectors that mediate the entry of a variety of molecules used in gene modulation, both in vivo and in vitro [68]. CPPs, also known as membrane translocation sequences, “Trojan peptides” or protein transduction domains (PTD) are peptide sequences with 5–30 amino acids, originated from a wide variety of sources. The CPP can efficiently enter cells through the cell membrane and also transports a wide range of macromolecules cargo for biological or therapeutic applications with low cytotoxicity or none. Transported cargoes can be plasmids, ONs, siRNAs, PNAs, peptides and proteins, drugs or liposome nanoparticles [78]. Depending on the concentration, direct translocation of CPP through cellular membrane and endocytosis are the principal mechanisms used to entry into the cell [10]. At low concentrations, the uptake is primarily endocytic but over a threshold direct translocation occurs. In the endosomal pathway, the CPP has the capacity to promote endosomal escape [23]. The formation of a stable complex between the CPP and the ON can be obtained either by the conjugation of the ON with the CPPs or due to the formation of noncovalent complexes between the two molecules [46]. According to their origin, there are three main classes of CPPs: peptides derived from proteins, chimeric peptides and synthetic CPPs, which present sequences usually designed based on structure–activity studies. On the other hand, considering their physical and chemical properties, three major classes of CPPs are recognized: cationic, amphipathic and hydrophobic [76]; some examples of each are shown in Table 1. Cationic CPPs are short amino acid sequences often consisting of arginine, lysine and histidine. These cationic amino acids mediate the interaction of the peptide with anionic/acidic motifs on the cell membrane. Arginine on CPP facilitates the internalization. Cationic arginine-rich peptides include the Tat peptide (86 aa) from the HIV transactivator protein TAT, which is an essential regulatory protein for HIV replication. Other examples include Penetratin, a 16 amino acid domain from the Antennapedia protein of Drosophila, a flock house virus (FHV) coat peptide (sequence 35–49) and oligoarginines [96]. Subsequently, conjugates of PMOs with arginine-rich CPPs, termed peptide-PMOs (PPMOs), were found to possess enhanced potency in vitro [117]. Conjugates coupled to R6-Penetratin (R6-Pen) improved serum stability and biological activity profile giving rise to a new class of PMO/PNA internalization peptides called Pips [49]. Amphipathic CPPs are chimeric peptides obtained by covalently attaching a hydrophobic domain to a NLS for efficient targeting to cell membranes. These peptides have hydrophilic blocks responsible for mediating translocation across the cell membrane. Amphipathic CPPs are divided into primary amphipathic CPPs, secondary amphipathic ␣-helical CPPs, ␤-sheet amphipathic CPPs and proline-rich amphipathic CPPs. C6M1 is an amphipathic peptide (18-mer) for siRNA delivery. This CPP has a reduced cytotoxicity and high solubility in aqueous solutions. C6M1 showed a random/helical structure in water or membrane-mimicking environments [50]. Hydrophobic peptides contain only hydrophobic amino acids and have a low net charge (less than 20% of the sequence).

Table 1 Peptides applied to modify splicing. Type Cationic

Amphipathic

Hydrophobic

Natriuretic and renal-protective peptides

Inhibitor peptides blocking protein–protein interaction

Peptide

Description

Action

Splicing

Ref.

GRKKRRQRRRPPQ

Carrier peptide

Improved exon skipping of dystrophin pre-mRNA

[43,113]

Penetratin

RQIKIWFQNRRMKWKK

Residues 48–60 of HIV-trans-activating transcriptional unit Antennapedia derived peptide

Carrier peptide

Splicing correction of the ␤-globin pre-mRNA

[65]

TP10

AGYLLGKINLKALAALAKKIL

Delivery agent

Splice correction assays

[11]

M918

MVTVLFRRLRIRRACGPPRVRV

Modified transportan molecule Derived from the tumor suppressor protein p14ARF

Cell penetrating peptide

Luciferase pre-mRNA splicing modulation

[30]

stearyl-(RxR)4

RXRRXRRXRRXR

Non-covalent delivery of splice-correcting oligos

Exon 23 skipping on splicing of human beta-globin pre-mRNA

[4]

PepFect 14

Stearyl-AGYLLGKLLOOLAAAALOOLL

Stearyl-oligo-arginine analog which has an ␣-aminohexanoic acid linker (x) between arginine residues. Derivative of TP-10

Cell penetrating peptide

Splice-correction activity in mdx mouse myotubes, a cell culture model of DMD.

[34]

GS

PLSRTLSVSS

CaMK II competitive inhibitor

Inhibit splicing and spliceosome assembly

CBD

LKKFNARRKLKGAILTTMLA

CaMK II autophosforilation inhibitor

MHRQEAVDCLKKFNARRKLKG

CaMK II autophosforilation substrate

MHRQETVDCLK

N-terminus of glycogen synthase Residues 290–309 of CaMK II, calmodulin binding region Residues 281–301, S286A, of CaMK II, autophosforilation region Residues 281–291 of CaMK II, autophosforilation region

BNP

SPKMVQGSGCFGRKMDRISSSS GLGCKVLRRH

Functional B-type natriuretic peptide

ASBNP

SPKMVQGSGCFGRKMDRISSSSGLGC KGKHPLPPRPPSPIPVCDTVRVTLG FVVSGNHTL

Alternatively spliced transcript of BNP resulting from the retention of intron 2

ASBNP.1

SPKMVQGSGCFGRKMDRISSSS GLGCKGKHPLPPRPPSPIPV

Carboxyl-terminal truncated form of the alternative isoform ASBNP

PL30-1 PL30-3 PL15-2 PL15-3 CD24-1

PYLFSCGEDKQVKCWDLEYNKVIRHYHGHL PQIITGSHDTTIRLWDLVAGKTRVTLTNHK DTTIRLWDLVAGKTR DLVAGKTRVTLTNHK HMTTEAKRAAKMEKKMKILLGGYQ

Synthetic peptides spanning the PLRG1 region that interacts with CDC5L Synthetic peptide of the CDC5L binding domain

CaMK II competes for calmodulin binding CaMK II inhibits autophosforilation

[85]

Inhibitor of splicing

CaMK II competitive inhibitor of autophosforilation This peptide has important autocrine, paracrine, and endocrine actions and has been used clinically in heart failure for its vasodilatory properties Maintains the folding structure of native BNP but lacks equipotency of BNP in stimulating cGMP in vascular cells and it also lacked the ability to vasorelax preconstricted arterial rings This form retained the effects of BNP on renal cells but lacked the effects on vascular cells and intact vascular rings PLRG1 peptides interact with CDC5L in vitro CDC5L peptide interacts with PLRG1 in vitro

Constitutive splicing

M.-M. Nancy et al. / Peptides 67 (2015) 1–11

Splicing inhibitor

Name HIV-1 Tat

Splicing isoform with inton retention

Short version of the splicing isoform

All peptides inhibit splicing in vitro by blocking the interaction between the spliceosome components CDC5L and PLRG1

[7]

5

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Hydrophobic CPPs could contain natural amino acids or can be chemically modified such as stapled peptides, prenylated peptides, and pepducins [76]. Recently, the modification of CPPs with hydrophobic moieties improves the interaction between the CPP and the ON, as shown in Fig. 2B [5,58,95]. Particularly, stearylation makes the peptide more hydrophobic, enhances condensation of the ON, the cytoplasmic and nuclear delivery, the adsorption at the cell surface, the cellular uptake, the translocation across the membrane without significant cytotoxicity and it also facilitates the endosomal escape [38]. The stearylation at the N-terminus of CPPs has been performed with stearyl-octaarginine (R8), stearyl-(RxR)4 and stearyl-TP10. For example, STR-R8 was applied for siRNA delivery; on the other hand, (RxR)4 is an oligoarginine analog capable of mediating delivery of SCOs to redirect pre-mRNA splicing in order to produce a functional protein [62]. CPP carriers as a powerful tool for splicing redirection The potential of CPP complexes as very promising tools in biotechnology and nanomedicine is rapidly increasing. CPPs have been applied for the transduction of different types of nucleic acids such as antisense and splice redirection ONs into mammalian cells both in vitro and in vivo [71]. Several novel CPPs have been shown to transfect and trigger specific biological response in a wide range of cell types without being cytotoxic [11,34]. Furthermore, in vivo experiments have revealed the capacity of CPP-mediated ONs to effectively inhibit tumor growth in tumor-bearing mice without triggering immune response, emphasizing even more the high therapeutic potential of CPP complexes [51]. In the particular case of splicing modulation, PNAs, PMOs, 2 -OMe or SSOs conjugated to CPPs like Tat, penetratin (Pen), and transportan (TP) can target specific genes and regulate gene expression (Table 1). The modulatory mechanism involves the interference with the process of exon recognition and the intron removal during splicing. An oligonucleotide directed to regions located at or close to a splice site can mask normal or aberrant splicing events leading either to exon exclusion or inclusion. The splice-oligonucleotides (SCOs) are used mainly to promote the named exon skipping, in which a specific exon with a mutation is skipped over, restoring the disrupted reading frame. This allows the generation of a functional protein [1] and was confirmed in HeLa cells for a mutated ␤-globin intron 2 carrying a cryptic splicing site [105]. Many ONs show an increased metabolic stability and affinity for the RNA target but without the activation of RNase H, with a steric-block mode of action useful for promoting therapeutic exon skipping [11,31]. CPP-aided splicing redirection is also a promising mechanism for in vivo and therapeutic applications. Antisense oligonucleotides such as PMO, PNA, SCOs or 2 -OMe have been useful for splicing redirection of pre-mRNA, which must be delivered into the cell nucleus conjugated to CPPs like Tat, TP10 or steryl-(RxR)4. The N-terminal stearylation of TP10 (STR-TP10) markedly improved the delivery efficiency of a phosphorothioate 2 -O-methyl RNA oligonucleotide (2 -OMe ON) for splice correction into cells [3]. Stearyl-(RxR)4 coupled with 2 -OMe SCOs promotes significant splice correction [40]. One of the most advanced examples in the development of approaches to modulate alternative splicing events as a therapeutic tool comprises the advances toward the treatment of the Duchenne muscular dystrophy (DMD) [27]. DMD is a sex-linked recessive disorder and is the most common and severe form of muscular dystrophy. There is no treatment for DMD, which is caused by mutations affecting the dystrophin gene of 2.6 Mb, which encodes a 14-kb mRNA comprising 79 exons [33]. The origin of DMD is a large family of mutations that generates a frame-shift resulting in a disfunctional dystrophin. Exon 51 skipping would be helpful

for 13% of DMD patients, including those with deletions of exons 50, 52, 45–50, 48–50 or 49–50. Exon 44 skipping could help 6% of DMD patients [18,32]. However, the use of ONs to remove the mutant exon and restore the proper reading frame could produce a truncated but partially functional dystrophin protein and potentially lead to milder symptoms. Several strategies using ONs have been tested in order to correct this disease. For example, in an mdx mouse model of DMD, it was described that systemic delivery of a PPMO-B targeted to exon 23 of the dystrophin gene induced high and sustained dystrophin expression in body-wide muscles, being particularly effective in heart, diaphragm, and quadriceps, the target muscles for the treatment of DMD [35,52]. The regular 1year administration of PMO targeting exon 23 rescued dystrophin expression in an animal model [121]. Moreover, the employment of an (R-Ahx-R)4AhxB–PMO conjugate at the neonatal stage of mdx mice, restored normal level of dystrophin expression in the diaphragm, where the protein was detected 22 weeks after the last dose of conjugate [79,116]. In a different experiment, conjugates of (RxR)4 to PMO or PNA, as PF14, a peptide derivative of Transportan 10 (TP-10) showed good splicing redirection for 2 OMe/PS oligonucleotides in exon skipping in mouse mdx muscle cells [49]. Another important target for dystrophic patients is myostatin, a negative regulator of skeletal muscle mass that may have important implications in disease conditions accompanied by muscle mass loss like DMD. Myostatin knockdown by exon 2 skipping is a potential therapeutic strategy to counteract muscle wasting conditions and dual myostatin and dystrophin skipping has potential as a therapy for DMD [55]. In this sense, antisense-mediated destructive exon skipping of myostatin has great potential as a combinatorial therapy alongside antisense mediated reframing and restoration of dystrophin expression in the treatment of DMD. The use of PMO conjugated to an arginine-rich cell-penetrating peptide (B-PMO) showed that B-PMO targeting myostatin is able to significantly increase muscle weight [69]. Importantly, exon skipping with PMOs and 2 -O-methyl phosphorothioate has been useful to induce dystrophin expression in DMD patients in phase II-III clinical trials, unfortunately this therapy failed to prevent disease progression when the disease was at an advanced stage [115]. The most advanced applications include three splice-switching ASOs which are in clinical trials phases II or III, all for the treatment of DMD. Prosensa has developed 2 -O-Me phosphorothioate oligonucleotides [42], while AVI BioPharma has favored the development of morpholino oligomers [2]. On the other hand, CPP-ON conjugates have been used in an immunodeficiency mouse model of Burkitt’s lymphoma, inhibiting tumor growth using a PNA targeted to an intronic sequence conjugated to a Lys-rich NLS peptide [17]. Some laminopathies are caused by mutations in the LMNA gene, which gives rise to at least three isoforms (lamin A, C, lamin A10) through alternative splicing. Lamin A and C are components of the fibrous nuclear lamina. Recently, it has been proposed the manipulation of LMNA splicing by splice-switching antisense oligonucleotides to correct the mis-splicing of LMNA at the transcript level [67]. In the past two decades, CPPs have been exploited as effective transfection agents for different types of cargoes, including nucleic acids. However, several CPP-based drugs are currently under preclinical and clinical evaluation. In this regard, although CPP translocation into cells was first discovered in the late 1980s [36,44], patents and clinical trials were not initiated until the mid 2000s (Table 2). Although it is still in development, there have been over 20 Phase I and Phase II clinical trials performed on over 2000 patients treated with CPP for a variety of indications [108]. Some of the biotech companies and academic institutions that have developed patents regarding CPP complexes to modulate splicing events are shown in Table 2 and include: Healthcare Biosciences

M.-M. Nancy et al. / Peptides 67 (2015) 1–11

7

Table 2 Patents regarding the use of CPPs to modulate splicing events. Patent

Inventors

Description

International application no. (date)

WIPOa Ref.

CHEMICALLY MODIFIED CELL-PENETRATING PEPTIDES FOR IMPROVED DELIVERY OF GENE MODULATING COMPOUNDS

Kariem Ahmed, Andaloussi Samir El, Peter Guterstam, Mattias HÄLLBRINK, Henrik Johansson, Ülo Langel, Taavi Lehto, Maria Lindgren, Imre Mäger, Rannar Sillard, Aizman Katri Rosenthal, Ulf Tedebark, Per Lundin Michael John Gait, Andrey Alexandrovich Arzumanov, Gabriela Dimitroval Ivanova (Medical Research Council) Ulf Tedebark (GE Healthcare Bio-Sciences)

A system for intracellular delivery of a cargo comprising at least one aliphatic component A which may contain several hetero atoms (N, S, O and P). Component A is attached to a CPP and/or a non-peptide analog

PCT/SE2009/051032 (16.09.2009)

WO2010039088

Cell penetrating peptides and conjugates of a CPP and a cargo molecule

PCT/GB2009/001261 (15.05.2009)

WO2009147368

System or reagent for intracellular delivery of a cargo, preferably a system for intracellular delivery of oligonucleotides. The system could be applied in diagnosis of diseases, as a research tool and as a targeting system CPP conjugates that include a cell penetrating peptide, a nucleic acid (oligonucleotide or siRNA), and a hydrophilic polymer (PEG) A construct comprising: a positively charged peptide; a targeting-delivery peptide; and the biologically active compound Antisense oligomer capable of binding to a selected target site in the human dystrophin gene to induce exon 44 skipping The use of an antisense compound for inducing exon inclusion as a treatment for Spinal Muscle Atrophy Improved compositions and methods for treating muscular dystrophy by using antisense molecules capable of binding to a selected target site in the human dystrophin gene to induce exon skipping A soluble human retrovirus HIV-1 glycoprotein gp41 trimer is a candidate for development of HIV-1 vaccines, diagnostics and therapeutics. The gp41 trimer is further attached to a CPP Antisense molecules capable of binding to a selected target site in the human dystrophin gene to induce exon skipping, and methods of use thereof to treat muscular dystrophy Compositions, methods and kits for the identification of a polypeptide that binds to a predetermined RNA sequence Compositions and methods for down-modulating the expression of splice variants of p53 for the treatment of disease

PCT/SE2011/050279 (22.09.2011)

WO2011115555

PCT/IB2006/003642 (20.06.2007)

WO2007069068

PCT/GB2009/001370 (03.12.2009)

WO2009144481

PCT/US2014/029689 (25.09.2014)

WO2014153220

PCT/US2012/067475 (06.06.2013)

WO2013082551

PCT/US2014/029610 (18.09.2014)

WO2014144978

PCT/IB2013/060453 (05.06.2014)

WO2014083518

PCT/US2009/061960 (29.04.2010)

WO2010048586

PCT/US2004/043401 (14.07.2005)

WO2005062947

WO2014011177 (16.01.2014)

PCT/US2012/046470

A cell-penetrating peptide or a construct comprising such a peptide of 15–25 amino acids comprising at least amino acids 1–14 of a mature mammalian tumor suppressor protein ARF

WO2008063113 (29.05.2008)

PCT/SE2007/001017

CELL PENETRATING PEPTIDES

SYSTEM FOR IMPROVED DELIVERY OF GENE MODULATING COMPOUNDS

CELL PENETRATING PEPTIDE CONJUGATES FOR DELIVERING OF NUCLEIC ACIDS INTO A CELL

Bertrand Alluis, Jean-Sebastien Fruchart (Diatos SA, France)

CONJUGATES FOR DELIVERY OF BIOLOGICALLY ACTIVE COMPOUNDS

Matthew Wood, Haifang Yin (Isis Innovation Limited)

EXON SKIPPING COMPOSITIONS FOR TREATING MUSCULAR DYSTROPHY

Richard K. Bestwick, Diane Elizabeth Frank (Sarepta Therapeutics, Inc).

INDUCED EXON INCLUSION IN SPINAL MUSCLE ATROPHY

Peter Linsley, Brian James Leppert (Sarepta Therapeutics, Inc). Edward M. Kaye (Sarepta Therapeutics, Inc.)

IMPROVED COMPOSITIONS FOR TREATING MUSCULAR DYSTROPHY

DESIGNING A SOLUBLE FULL-LENGTH HIV-1 GP41 TRIMER

Venigalla B. Rao, Guofen Gao (The Catholic University of America)

MULTIPLE EXON SKIPPING COMPOSITIONS FOR DMD

Peter Sazani, Ryszard Kole (Sarepta Therapeutics, Inc.)

METHODS AND COMPOSITIONS FOR IDENTIFYING RNA BINDING PROTEIN ANTISENSE P53 PHOSPHORODIAMIDATE MORPHOLINO COMPOSITITONS, METHODS AND INDICATIONS CELL-PENETRATING PEPTIDES AND CONSTRUCTS CONTAINING THEM CONSISTING 15–25 AMINO ACIDS OF TUMOR SUPRESSOR PROTEIN P14ARF OR P19ARF

James Eberwine, Ulo Langel, Kalle Kilk, Jennifer Zielinski (Univ Pennsylvania)

a

Larry J. Smith, Patrick L. Iversen (Smith Holdings, Llc, Eleos, Inc.)

Samir El-Andaloussi, Henrik Johansson, Uelo Langel

WIPO: World Intellectual Property Organization.

(Sweden), the Medical Research Councilin (Great Britain), Sarepta Therapeutics Inc. (USA), Diatos SA (France), The Catholic University of Amerca (USA) and the University of Pensylvania (USA). Importantly, this CPP delivery has been reported to be safe for the patient

given that none of the clinical trials has reported any adverse effect. Given the recent biotechnological advances, these clinical data could guide the development of macromolecular therapeutics in the upcoming years.

8

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Splicing arrest using peptide inhibitors of protein kinases As previously mentioned, the spliceosome consists of at least 150 proteins involved in splicing catalysis. Interestingly, some of them play an important role as protein kinases, given that phosphorylation and dephosphorylation cycles may be required for splicing. Several mammalian kinases have been implicated in splicing regulation, like PP1␥ and PP2A, but also other phophatases with similar specificities [75,114]. As an example, phosphorylation and dephosphorylation of the snRNP U1-70K is an important regulatory switch for splicing which is controlled by kinase activity [77,107] and the same occurs for SF2/ASF [19]. Particularly, the splicing regulators corresponding to the SR family of proteins have kinase motifs in order to phosphorylate other members of the SR family including SRPK1 and SRPK2, Clk/Sty [21,45,60,109], DNA topoisomerase I [93], cyclin E–cdk2 [97] and CaMK II-like kinase [86]. For CaMK II-like kinase, Parker and Steitz demonstrate the inhibitory effect of four peptides that were able to block the mammalian spliceosome assembly and pre-mRNA splicing in vitro. This four peptides were previously described as inhibitors of CaMK II via distinct mechanisms: the glycogen synthase (GS) fragment [89], the calmodulin binding domain (CBD) peptide [88,120], the CaMK II autophosphorylation inhibitor [100] and the CaMK II autophosphorylation substrate [118]. Regarding splicing, GS and CBD block the assembly of spliceosomal complex C, while Calmodulin kinase II-directed peptides inhibited pre-mRNA splicing. These observations suggested that a protein similar to CaMK II might be involved in spliceosome assembly and splicing [101]. The study mentioned above supports the notion that peptide inhibitors offer a potentially more selective approach to study the role of protein phosphorylation in pre-mRNA splicing as a tool to study spliceosome structure and function. More importantly, these observations led to other studies directed to develop new tools using small molecules designed to block splicing with high specificity, which can be applied as new drugs for different applications. Interestingly, these inhibitory peptides have been the origin to develop therapeutic tools applied to correct aberrations in splicing. Some of the applications based on kinase inhibitors include anti-HIV drugs [12], antitumor compounds [90], small-molecule inhibitors of histone acetyltransferases and histone deacetylases [59] and the specific inhibitor of splicing named spliceostatin and derivatives [9,83]. Future efforts will still be necessary toward the complete optimization of these compounds as potential human therapeutics. Natural protective peptides that modulate alternative splicing The B-type natriuretic peptide (BNP) is an important clinical and therapeutic peptide called nesiritide, which is used in the diagnosis and treatment in patients with heart failure for its vasodilator properties [91]. BNP is generated from a small multiexonic gene and although it was originally identified in porcine brain extracts [104], it is expressed mainly in the cardiac ventricles [82]. The mature form of the peptides contains short carboxyl and amino termini and a central 17-aa ring. BNP has important autocrine, paracrine, and endocrine actions that are mediated through a specific receptor that involves the activation of cGMP [39]. BNP, like the hormone secreted from mammalian atria ANP (atrial natriuretic peptide), regulates the homeostatic balance of body fluid and blood pressure and is expressed as a prepro-hormone that is processed to a mature form of 32 aa [119]. However, the use of BNP in clinical treatment has been limited due to hypotension and was associated with worsening renal function [94]. As an alternative, an investigation into the complexity of the human proteome provided a peptide that might be used in therapeutics and recently an alternatively spliced transcript of BNP

resulting from the retention of intron 2 in failing human left ventricular tissue was identified [84]. This transcript generates a 60 aa-long peptide that maintains the structure of native BNP. This alternatively spliced transcript is expressed 2log-fold less than BNP and it has been found in human cardiac myocytes using specific antiserum. ASBNP, as they named this alternatively spliced isoform, was synthesized and resulted less potent than BNP in stimulating cGMP in vascular cells and it also lacked the vasorelaxation effect over preconstricted arterial rings. Considering the structural properties of BNP and based in the interest to develop new therapeutic peptides, a carboxyl-terminal truncated form of the alternatively spliced isoform was designed, which was called ASBNP.1. This isoform retained the protective effects of BNP on renal cells but lacked the vascular effects of BNP in relation to the limits observed with the complete peptide regarding hypotension and renal functions. ASBNP.1 enhanced the glomerular filtration rate (GFR), which is important for those cardiorenal syndrome patients in whom the GFR enhancement might be achieved without risk of hypotension. These studies showed the potential of natural splicing-derived peptides in the treatment of specific diseases. Peptides blocking protein–protein interaction stall the spliceosome. CDC5L and PLRG1 are two nuclear proteins that are highly conserved across species that have been identified as components of the human spliceosome [92,122]. CDC5L is able to bind to different proteins, including PLRG1 in order to form an active complex needed for splicing [6]. Apart from their role in splicing, several other cellular functions have been attributed to both the CDC5L and PLRG1 proteins. For example, it has been observed that overexpression of CDC5L shortens the G2 phase in the cell cycle and reduces cell size, suggesting that it has a role in regulating G2 progression and mitotic entry [16]. Moreover, genome-wide gene expression analysis reveals that CDC5L modulates the expression of a set of genes involved in the mitosis and the DNA damage response, suggesting that CDC5L is a key regulator of mitotic progression [81]. It has also been shown that the N-terminal domain of CDC5L is capable of activating transcription [63]. On the other hand, PRL1 has been shown to regulate cell proliferation and apoptosis [57]. All these findings highlight the potential of CDC5L and PRL1 as targets for cancer therapy. Given the attractive cellular roles of the splicing factors CDC5L and PLRG1, a detailed analysis of the interaction domain of both proteins was performed by using short peptides derived from sites surrounding phylogenetic highly conserved amino acids. Initially, the binding region was identified both in CDC5L and PLRG1 [8]. Over this region, 25mer–30mer synthetic peptides were made and they were added to splicing reactions as competitors to the endogenous protein. Those peptides that were able to inhibit splicing were considered to be essential for the protein binding and complex formation. Efficient inhibition of the active peptides was confirmed using HeLa nuclear extracts and bacterially expressed and purified protein with different pre-mRNA substrates. Furthermore, pull-down experiments also confirmed the specific activity for the chosen peptides [7]. While studying in detail the mechanism underlying the splicing inhibition mediated by the active peptides, it was more likely that they do not prevent the spliceosome assembly but instead inhibit splicing by interfering with essential contacts between the CDC5L and PLRG1 proteins. The effective use of small peptides as splicing inhibitors could be applied not only for the regions described above which are essential for the interaction between CDC5L and PLRG1, but it can also be extended to other regions involved in protein–protein interactions for several splicing factors. According to these observations,

M.-M. Nancy et al. / Peptides 67 (2015) 1–11

peptides could be potentially very useful tools in determining the functions of interacting proteins in the splicing mechanism and may facilitate the future development of splicing inhibitors that can be used to specifically block a given pre-mRNA splicing event. Conclusions and perspectives Alternative splicing has rapidly emerged as a new drug target. Given the importance of alternative splicing, it becomes logical that low-molecular-mass compounds that inhibit certain splicing events or stages in the splicing reaction have been identified. However, the specificity of these compounds remains a problem. During the past years, research focused on changing incorrect splice-site selection to the appropriate function led to the identification of several components with the ability to affect alternative splicing decisions. ASOs have been used to restore gene function in genetic disorders by correcting aberrant splicing caused by mutations, removing nonsense mutations from mRNA by exon skipping, or regulating alternative splicing process. Systemic delivery is one of the major obstacles to the successful application of ON-based therapies. To enhance the efficiency of systemic delivery of AMOs, new carrier systems, such as CPPs are being developed. CPPs function as potent vehicles for splice correction, which could be used to target aberrant splice sites occurring in several genetic disorders and cancers. Besides their application as carriers, natural and synthetic peptides are a real possibility for molecules that will directly interact with the spliceosome factors or that will indirectly alter molecular pathways that regulate splice-site selection. The lack of toxicity of peptidic molecules, even when they are used in high micromolar concentrations makes peptides an interesting candidate for current and future therapies. Finding substances that possess not only the ability to regulate splicing, but that could also show specificity for a certain step or event of splicing still represents a challenge. Future developments in this matter will allow us to treat human diseases caused by anomalous splicing. Further improvements on the peptidic molecules described here, will expand the repertoire of therapeutic molecules and benefit additional patients. Acknowledgments This study was elaborated within the CONACYT funding (CONACYT-CB2009-01/0133317). R. Martínez-Contreras was the responsible of the funding. N. Martínez-Montiel is recipient of the CONACYT scholarship for graduate studies. References [1] Aartsma-Rus A, van Ommen GJ. Antisense-mediated exon skipping: a versatile tool with therapeutic and research applications. RNA 2007;13:1609–24. [2] Aartsma-Rus A, van Ommen GJ. Less is more: therapeutic exon skipping for Duchenne muscular dystrophy. Lancet Neurol 2009;8:873–5. [3] Abes R, Moulton HM, Clair P, Yang ST, Abes S, Melikov K, et al. Delivery of steric block morpholino oligomers by (R-X-R)4 peptides: structure-activity studies. Nucleic Acids Res 2008;36:6343–54. [4] Abes S, Moulton HM, Clair P, Prevot P, Youngblood DS, Wu RP, et al. Vectorization of morpholino oligomers by the (R-Ahx-R)4 peptide allows efficient splicing correction in the absence of endosomolytic agents. J Control Release 2006;116:304–13. [5] Abes S, Turner JJ, Ivanova GD, Owen D, Williams D, Arzumanov A, et al. Efficient splicing correction by PNA conjugation to an R6-Penetratin delivery peptide. Nucleic Acids Res 2007;35:4495–502. [6] Ajuh P, Kuster B, Panov K, Zomerdijk JC, Mann M, Lamond AI. Functional analysis of the human CDC5L complex and identification of its components by mass spectrometry. EMBO J 2000;19:6569–81. [7] Ajuh P, Lamond AI. Identification of peptide inhibitors of pre-mRNA splicing derived from the essential interaction domains of CDC5L and PLRG1. Nucleic Acids Res 2003;31:6104–16. [8] Ajuh P, Sleeman J, Chusainow J, Lamond AI. A direct interaction between the carboxyl-terminal region of CDC5L and the WD40 domain of PLRG1 is essential for pre-mRNA splicing. J Biol Chem 2001;276:42370–81.

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