Patent Publication Number: US-2023158166-A1

Title: Biologically active compounds

Description:
RELATED APPLICATION 
     This application is a continuation of U.S. application Ser. No. 16/061,548 filed Jun. 12, 2018, which issued as U.S. Pat. No. 11,541,124, which is a national stage filing under 35 U.S.C. § 371 of International Patent Application Serial No. PCT/GB2016/054028, filed Dec. 21, 2016, which claims the benefit of priority to UK Application No. 1522548.5 filed Dec. 21, 2015, the contents of which is incorporated herein by reference in its entirety. 
    
    
     SEQUENCE STATEMENT 
     This application contains a Sequence Listing which has been submitted in XML format via EFS-Web and is hereby incorporated by reference in its entirety. Said XML copy, created on Dec. 16, 2022, is named Y9899_01002 SL.xml and is 418,510 bytes in size. 
     FIELD OF THE INVENTION 
     The present invention relates to improvements in drug delivery. 
     More particularly it relates to the use of Cell Penetrating Agents (CPA&#39;s), and more particularly still to the use of Cell Penetrating Peptides (CPP&#39;s) which have been stabilized by, for example: i) stapling two amino acids to form Stapled CPP&#39;s (StaP&#39;s) or ii) stitching three or more amino acids to form stitched CPP&#39;s (StiP&#39;s). 
     These stabilized CPP&#39;s are conjugated to a drug or Biologically Active Compound (BAC) directly or via a Bi-Functional Linker (BFL) so that the BAC can be carried though a cell membrane by the CPP. The resulting molecules are referred to as Drug Carrying Cell Penetrating Molecules (DCCPM&#39;s). 
     The preferred BAC&#39;s delivered in this manner are oligonucleotides (ON&#39;s), more preferably still electrically low charge carrying oligonucleotides (charge −3 to +3 at pH 7.5) and most preferably electrically neutral oligonucleotides (charge −1 to +1 at pH 7.5), such as, but not limited to, polynucleic acids (PNAs), phosphorodiamidate morpholino oligonucleotides (PMO&#39;s) or modified derivatives thereof. 
     The preferred BFL may be PEGylated, comprising poly ethylene glycol (PEG) groups including modifications such as an amine group, or incorporate a spacer, such as 13-Ala. These modifications can improve solubilisation or provide appropriate spacing between functional moieties. 
     The invention also relates to a method of facilitating the uptake of a BAC into a cell, the use of a DCCPM in the treatment of a disease requiring alteration of an endogenous or exogenous gene, a method of improving the bioavailability of a drug or BAC, a method of introducing a drug or BAC to a site which is refractory to the drug or BAC in its native state, a method of treating a subject comprising administering the DCCPM&#39;s of the invention and to a pharmaceutical composition comprising the DCCPM and one or more pharmaceutically acceptable excipients. 
     Still further aspects will be apparent from the detailed description. 
     BACKGROUND TO THE INVENTION 
     n the treatment of all diseases it is desirable to deliver a drug or BAC into the body, and more preferably into a cell, at a target site, in a manner that ensures a maximal effect with minimal toxicity. This can be challenging. 
     An example of drugs or BACs which are delivered in a targeted manner are oligonucleotides (ON&#39;s), which term includes ON analogues. 
     ON&#39;s can target essential DNA, RNA and protein sequences and can modulate gene expression in a number of ways that includes steric blocking to suppress (i) RNA splicing, (ii) protein translation or (iii) other nucleic acid:nucleic acid or nucleic acid:protein interactions. 
     Specifically, the hybridisation of ON&#39;s to specific RNA sequence motifs prevents correct assembly of the spliceosome, so that it is unable to recognise the target exon(s) in the pre-mRNA and hence excludes these exon in the mature gene transcript. Exclusion of an in-frame exon can lead to a truncated yet functional gene product; exclusion of an out of frame exon results in a frame-shift of the transcript, potentially leading to a premature stop codon and a reduction in the target gene expression level. 
     Additionally, ON&#39;s can be designed to target 5′ translation initiation start sites of viral gene transcript(s) to prevent binding of the translational machinery. Using antisense oligonucleotides (ASO) to suppress viral translation is a well-established technology 1  and has progressed into clinical trials for viral haemorrhagic fevers such as Marburg and Ebola 2,3 . 
     Also, ON&#39;s can be designed to form aptamers such that the secondary and tertiary structures can bind proteins or other cellular targets thus impacting on specific gene expression levels or other cellular processes (e.g. post-translational modifications). 
     An advantage of steric blocking based suppression over that of siRNA/RNAi based RNase H-induction of the RNA Induced Silencing Complex is the reduced likelihood of off target side effects. 
     Modifications of an ON to produce a negatively charged backbone improve stability 4-7 , but these backbone chemistries e.g. 2′O-Methyl Phosphothioate analogues, elicit membrane toxicity issues, cause thrombocytopaenia and injection site problems upon clinical translation 8 , such that efficacy is prevented by toxicity issues, even when administration protocols become increasingly intermittent 9 . 
     Indeed WO2013/150338 and WO 2014/053622 both disclose delivering negatively charged ON&#39;s of small size (typically smaller than 1.5 KDa) by complexing them with positively charged linear or stapled peptides of equal or greater than 15 amino acids and in the range of 15-27 amino acids. 
     JACS, Vol 136, 2014, GJ Hilnski et al, describe stapled and stitched peptides that are able to penetrate cells. Reference is made to the possibility that these peptides could be used to deliver an oligonucleotide, presumably in the same manner as disclosed in the international applications disclosed above, i.e. by complexation. There is nothing to suggest creating new entities of much larger size (greater than 1.5 KDa, through 2.5 KDa, 5 KDa, 7.5 KDa, 10 KDa, 12.5 KDa or more) by covalently linking a BAC with a CPA, optionally via a BFL and indeed, the prior methodology requires the respective components to have opposite charges to facilitate complexing. 
     The use of electrically low charge carrying oligonucleotides (charge −3 to +3 at pH 7.5) and most preferably electrically neutral oligonucleotides (charge −1 to +1 at pH 7.5), such as, but not limited to, polynucleic acids (PNAs), phosphorodiamidate morpholino oligonucleotides (PMO&#39;s), (covalently) conjugated directly or indirectly, using a BFL, was not apparent and indeed, limiting the charge on the ON further allows the use of smaller peptides (less than 15 amino acids in length, through 14, 13, 12, 11, 10, 9, 8, 7, 6 to as few as 4 or 5) as carriers. 
     The use of uncharged ON backbones, such as phosphorodiamidate morpholino oligonucleotides (PMOs), represent attractive BAC&#39;s as they have an impeccable safety record in a preclinical and clinical setting. 2,10-13    
     However, their ability to penetrate cells and access their targets is compromised due to their uncharged nature 14 . 
     Overcoming the problem of facilitating their entry into cells is therefore desirable. 
     Over the last 20 years much research has been dedicated to developing CPA&#39;s that facilitate delivery of drugs and BAC&#39;s to the biological site of action. 
     The approach has generally been to use charged peptides as non-covalent complexes to facilitate cell entry of a BAC. Conjugation has also been tried. 
     WO2014/064258 is an example of the existing conjugating art. A negatively charged ON is coupled to a targeting peptide via a linker. The targeting peptide is a receptor targeting moiety, and not a stapled or stitched peptide, and indeed considerable doubt exists as to whether DNA or RNA molecules can gain cell entry using a receptor targeting moiety as once a negatively charged ON is bound to such a moiety, non-covalent interactions alter its conformation 15 . 
     WO89/03849 discloses oligonucleotide-polyamide conjugates. There is no disclosure of the use of stitched or stapled peptides. The methodology described uses oligonucleotides as a scaffold for the chain extension of peptides and not as a conjugate for delivery, per se. 
     WO2011/131693 describes nucleic acid constructs which contain a nucleic acid specific for a given target gene and a selective inhibitor of a neurotransmitter transporter. There is no disclosure of the use of stitched or stapled peptides as a delivery agent. 
     A peptide capable of effecting peptide-mediated cell delivery may also be referred to as a Cell Delivery Peptide (CDP). Examples include: poly arginine, penetratin (based upon an antennapedia homeodomain), or PMO internalization peptides (PIPs). 
     However, since their first description 16  and given that many CPPs contain multiple arginines, β-alanine and 6-aminohexanoic acid residues, (e.g. poly-Arg12, TAT, Penetratin, Pip6a) [database maintained at crdd.osdd.net/raghava/cppsite/] 17 , it is surprising that no CPP-delivered drug has progressed through all phases of clinical trials. In part, this may be because the common arginine-rich core, which makes most CPP&#39;s effective, also causes membrane deformities 18  and in higher mammals this manifests as prohibitive toxic side effects, such as tubular degeneration of the kidney 19 . 
     At a physiological pH, and based on pKa of amino acid R groups, a formal charge (FC) can be calculated based on the formula: 
     
       
         
           
             FC 
             = 
             
               V 
               - 
               N 
               - 
               
                 B 
                 2 
               
             
           
         
       
     
     Where, V=valence electrons of the neutral atom in isolation; N=the number of non-bonding valence electrons on the defined atom; B=the total number of electrons shared in bonds. 
     Indeed, based on this, the CPPs typically used to date harbour many positively charged residues. It has been shown that there is a correlation between this positive charge and membrane toxicity 20 . 
     Therefore, CPPs with a lower amount of positively charged residues within the amino acid sequence, whilst retaining the ability to cross a biological membrane, will be more clinically relevant. 
     The Applicant has overcome this major impediment by utilising stabilized CPA&#39;s. By linking a drug or BAC to a stabilized CPA, including stitched and stapled peptides, they have surprisingly obtained enhanced cellular uptake dynamics, 10-20 fold better than current state of the art for CPA&#39;s 21,22.    
     They have illustrated this by delivering an ON targeted to repair a gene producing dystrophin. Targeting specific genes with ON is of course in itself known, as illustrated by, for example, WO2009/054725 and WO2010/123369. These publications however use a negatively charged backbone and deliver the cargo directly or using complexation. 
     One way to prepare stapled and stitched peptides, two linked amino acids (stapled) or three or more linked amino acids (stitched), is to incorporate amino acids into the peptide that are modified to bear e.g. an olefin (alkene) group (which may be incorporated at defined relative positions during solid-phase peptide synthesis). For example, on-resin ring-closing metathesis is then used to close one (stapled [denoted as StaP herein]) or two or more (stitched [denoted as StiP herein]) all-hydrocarbon cross-links that induce the peptide to adopt a stabilised structure, typically, but not essentially an alpha helix. For StaP&#39;s, it is preferred to use either one or both enantiomers of the un-natural amino acids, termed the S5 (S-pentenylalanine) or R5 (R-pentenylalanine), or the S8 (S-octenylalanine) or R8 (R-octenylalanine), depending on the stereo-chemical configuration. For StiP&#39;s, a further un-natural olefin-bearing α, α-di-substituted amino acid (B5 or B8) is utilised. Cross linking strategies are however not restricted to ring-closing metathesis of un-natural olefin-bearing a, α-di-substituted amino acids. Other cross-linking chemistry&#39;s may be used to stabilize the peptide, such as ring-closing metathesis between O-allylserine analogues (S-OAS or R-OAS). 
     The cellular entry dynamics of existing CPAs and the StiP&#39;s and StaP&#39;s differ. Traditional CPPs enter cells via energy-independent direct plasma membrane translocation or via energy-dependent, clathrin and caveolin-mediated endocytosis; whereas the StiP&#39;s and StaP&#39;s utilised in the invention enter via an energy dependent, but clathrin and caveolin independent mechanism 21,23 . Given that StiP&#39;s and StaP&#39;s uptake is abrogated with reduced cellular decoration of heparin sulphate 21  a macropinocytotic entry mechanism is infered 24 , suggesting this altered entry mechanism enables enhanced cellular uptake and bio-distribution compared to the state of the art. 
     Relative to their unmodified peptide precursors, StaP&#39;s and StiP&#39;s generally exhibit robust cellular uptake, significant resistance to proteolytic degradation, and in vivo stability that can support a half-life of more than 12 hours in non-human primates 25 . It is likely that this increase in drug-likeness stems from the highly rigidified structure and the burial of the backbone amide bonds in the core of e.g. the α-helix. This structural rigidity also decreases the likelihood that StiP&#39;s and StaP&#39;s will be immunogenic, as the design of major histocompatibility complexes is such that peptides must adopt an extended conformation to be presented. The potential reduced or lack of membrane toxicity and immunogenicity enhances the clinical translatability of compounds when conjugated to drugs and BAC&#39;s such as ON&#39;s. 
     The BAC and CPP can be covalently conjugated directly, or covalently conjugated via a BFL. Many functional groups may be used for conjugation reactions. 
     ONs can be used to induce a steric block to any gene in humans, animals and lower order organisms and thus can be applied to natural disease (including genetic and age-related diseases) or acquired diseases in humans and animals. 
     For example, viral haemorrhagic fevers (VHFs) are animal-borne illnesses in which a prolonged inflammatory cytokine response leads to the gradual destruction of veins and arteries. Causes of VHF include Ebola and Marburg viruses and several Arena viruses; these diseases are presently considered untreatable. Viral haemorrhagic fevers are characterized by high fever and bleeding disorders, and can cause death by shock and organ failure. ASOs can be designed to target 5′ translation initiation start sites of viral gene transcript(s) to prevent binding of the translational machinery. Using ASO to suppress viral translation is a well-established technology 1  and has progressed into clinical trials for viral haemorrhagic fevers such as Marburg and Ebola 2,3 . One PMO, AVI-7537 was evaluated for human use in the West African Ebola outbreak in 2014-15. 
     Some tissues are particularly refractory to naked PMO transfection, e.g. heart, which may reflect differential vesicle-mediated PMO uptake mechanisms 23 . In fact, direct intra-cardiac injection of naked PMO does not even lead to efficient transfection 26 , and refractory tissues tend to require repeat administration or high dose strategies 27-29 . However, whilst CPP conjugation improves PMO bio-distribution and serum stability 30-32 , toxicity is still a major roadblock for pipeline development 19 . 
     For effective clinical translation of steric blocking ASOs, CPPs need to effectively deliver the BAC to either the cytoplasm or nucleoplasm whilst limiting any toxicity associated with cell entry. 
     Thus, providing DCCPM&#39;s which are able to deliver a drug or BAC more efficiently or to a target site, or with lower toxicity and immunogenicity would be highly desirable. 
     BRIEF SUMMARY OF THE DISCLOSURE 
     In accordance with a first aspect of the present invention there is provided a drug carrying cell penetrating molecule (DCCPM) comprising:
         i. a biologically active compound (BAC), and   ii. a cell penetrating agent (CPA), which BAC and CPA are linked directly or via a bi-functional linker (BFL),   and wherein the CPA is a stabilized peptide (CPP) which has a conformation imposed upon it by stapling to form a stapled peptide (StaP) or stitching to form a stitched peptide (StiP).       

     A stapled peptide (StaP) may be formed by, for example, stapling two conformationally adjacent amino acids together, and a stitched peptide (StiP) may be formed by, for example, stitching at least three conformationally adjacent amino acids to form a stitched peptide (StiP). 
     The stapling or stitching results in the formation of a cross link or bridge between two conformationally adjacent amino acids of the peptide. 
     In a preferred embodiment the cross link or bridge comprises two components, a hydrocarbon bridge and a terminal methyl group. The hydrocarbon bridge may be composed of a double hydrocarbon bond or a single hydrocarbon bond. 
     The CPP preferably comprises at least two un-natural amino acids bearing all-hydrocarbon tethers (e.g. α-methyl,α-pentenyl glycine). 
     The preferred stapled or stitched CPPs incorporate one or more of: a (S)-pentenylalanine (S5) or its enantiomer (R5), a S-octenylalanine (S8) or its enantiomer (R8) or combinations thereof (e.g R-octenylalanine/S-pentenylalanine (R8/S5) or S-octenylalanine/R-pentenylalanine (S8/R5). 
     The preferred unnatural amino acids incorporated into the CPPs and reacted to form a cross link or bridge between them are illustrated in Table 1 and some exemplary and preferred resulting CPPs are illustrated in Table 2. 
     
       
         
           
               
             
               
                 TABLE 1 
               
               
                   
               
               
                 Unnatural Amino Acids 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
             
               
                 TABLE 2 
               
               
                   
               
               
                 SEQ ID 
                   
                   
                   
               
               
                 NO: 
                 Peptide Sequence 
                 Length 
                 Peptide Type 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                 3 
                 TRQARRNRRRRWRRAAAA 
                 18 
                 Non RCM 
               
               
                   
               
               
                 4 
                 TRQARRNRRRRWRERQR 
                 17 
                 Non RCM 
               
               
                   
               
               
                 5 
                 RQIKIWFQNRRMKWKK 
                 16 
                 Non RCM 
               
               
                   
               
               
                 6 
                 RRRRRRRRWRRR 
                 12 
                 Non RCM 
               
               
                   
               
               
                 7 
                 LSQETFSDLWKLLPEN 
                 16 
                 Non RCM 
               
               
                   
               
               
                 8 
                 NQLKRSFFALRDQI 
                 14 
                 Non RCM 
               
               
                   
               
               
                 8 
                 NQLKRSFFALRDQI 
                 14 
                 Non RCM 
               
               
                   
               
               
                 9 
                 TILKASVDYIRKLQREQQRAKEL 
                 23 
                 Non RCM 
               
               
                   
               
               
                 6 
                 RRRRRRRRWRRR 
                 12 
                 Non RCM 
               
               
                   
               
               
                 10 
                 RRRRRRRRRRRR 
                 12 
                 Non RCM 
               
               
                   
               
               
                 11 
                 YGRKKRRQRRRP 
                 12 
                 Non RCM 
               
               
                   
               
               
                 12 
                 RKFKRLFQ 
                 8 
                 Non RCM 
               
               
                   
               
               
                 13 
                 NELKRSFFALRDQI 
                 14 
                 Non RCM 
               
               
                   
               
               
                 14 
                 NQL-R8-RS-FFAL-S5-DQI 
                 14 
                 Non RCM 
               
               
                   
               
               
                 15 
                 KNHTHQQDI 
                 9 
                 Non RCM 
               
               
                   
               
               
                 16 
                 NELKRSFFALRDQIPSLQGEKASRAQILDKA 
                 42 
                 Non RCM 
               
               
                   
                 TEYIQYNLRRK 
                   
                   
               
               
                   
               
               
                 17 
                 KATEYIQYNLRRKNHTHQQDIDDL 
                 24 
                 Non RCM 
               
               
                   
               
               
                 18 
                 ASTLFETFYLGGLLG 
                 15 
                 Non RCM 
               
               
                   
               
               
                 19 
                 RRGSRPSGA-S5-RRR-S5-R 
                 15 
                 Non RCM 
               
               
                   
               
               
                 20 
                 FNINDRIKELGTLI 
                 14 
                 Non RCM 
               
               
                   
               
               
                 21 
                 DHIKDSFHSLRDSVPSLQGEKASRAQILDK 
                 42 
                 Non RCM 
               
               
                   
                 ATEYIQYNLRRK 
                   
                   
               
               
                   
               
               
                 22 
                 EYIQYNLRKNHTHQQDIDDLKRQNALLEQQ 
                 36 
                 Non RCM 
               
               
                   
                 VRALGG 
                   
                   
               
               
                   
               
               
                 23 
                 SSLFERFYNLVTPAGG 
                 16 
                 Non RCM 
               
               
                   
               
               
                 24 
                 NSSFADFFHTVPYNLL 
                 16 
                 Non RCM 
               
               
                   
               
               
                 25 
                 TRQARRN-S5-RRR-S5-RR 
                 14 
                 Non RCM 
               
               
                   
               
               
                 26 
                 RRGSRPSGA-S5-RRR-S5-RAAAA 
                 19 
                 i, i + 4 Staple 
               
               
                   
               
               
                 27 
                 S5-RRQ-S5-RRDRQRRRRR 
                 15 
                 i, i + 4 Staple 
               
               
                   
               
               
                 28 
                 TRQ-S5-RRQ-S5-RRRWRERQR 
                 17 
                 i, i + 4 Staple 
               
               
                   
               
               
                 29 
                 SEELV-S5-EAH-S5-LCTLLENAIQDTVREQ 
                 26 
                 i, i + 4 Staple 
               
               
                   
               
               
                 30 
                 SEELVAEAH-S5-LCT-S5-LENAIQDTVREQ 
                 26 
                 i, i + 4 Staple 
               
               
                   
               
               
                 31 
                 SEELVAEAHNLCTLLE-S5-AIQ-S5-TVREQ 
                 26 
                 i, i + 4 Staple 
               
               
                   
               
               
                 32 
                 DRRQRRR-S5-RQR-S5-RRR 
                 15 
                 i, i + 4 Staple 
               
               
                   
               
               
                 33 
                 S5-RRQ-S5-RRRRQRRRRR 
                 15 
                 i, i + 4 Staple 
               
               
                   
               
               
                 34 
                 S-S5-ELV-S5-EAHNLCTLLENAIQDTVREQ 
                 26 
                 i, i + 4 Staple 
               
               
                   
               
               
                 35 
                 SEELVAEA-S5-NLC-S5-LLENAIQDTVREQ 
                 26 
                 i, i + 4 Staple 
               
               
                   
               
               
                 36 
                 SEELVAEAHNLC-S5-LLE-S5-AIQDTVREQ 
                 26 
                 i, i + 4 Staple 
               
               
                   
               
               
                 37 
                 SEELVAEAHNLCTLLENAI-S5-DTV-S5-EQ 
                 26 
                 i, i + 4 Staple 
               
               
                   
               
               
                 38 
                 FS-S5-LWK-S5-L 
                 8 
                 i, i + 4 Staple 
               
               
                   
               
               
                 39 
                 FM-S5-YWK-S5-L 
                 8 
                 i, i + 4 Staple 
               
               
                   
               
               
                 40 
                 QTFS-S5-LWK-S5-L 
                 10 
                 i, i + 4 Staple 
               
               
                   
               
               
                 41 
                 PPKKFR-S5-LFF-S5-S 
                 12 
                 i, i + 4 Staple 
               
               
                   
               
               
                 42 
                 KK-pff-R-S5-LFF-S5-S 
                 10 
                 i, i + 4 Staple 
               
               
                   
               
               
                 43 
                 RK-pff-S5-RLF-S5-SY 
                 10 
                 i, i + 4 Staple 
               
               
                   
               
               
                 44 
                 RKF-S5-RLF-S5-SY 
                 10 
                 i, i + 4 Staple 
               
               
                   
               
               
                 45 
                 R-pff-K-S5-RLF-S5-SY 
                 10 
                 i, i + 4 Staple 
               
               
                   
               
               
                 46 
                 AM-S5-YVVK-S5-L 
                 8 
                 i, i + 4 Staple 
               
               
                   
               
               
                 47 
                 QTFSD-R5-WK-S5-L 
                 10 
                 i, i + 4 Staple 
               
               
                   
               
               
                 48 
                 KKFR-S5-LFF-S5-S 
                 10 
                 i, i + 4 Staple 
               
               
                   
               
               
                 49 
                 RRLFR-S5-NLFL-S5-T 
                 12 
                 i, i + 4 Staple 
               
               
                   
               
               
                 50 
                 RR-pff-S5-RLF-S5-SY 
                 10 
                 i, i + 4 Staple 
               
               
                   
               
               
                 51 
                 RKA-S5-RLF-S5-SY 
                 10 
                 i, i + 4 Staple 
               
               
                   
               
               
                 43 
                 RK-pff-S5-RLF-S5-SY 
                 10 
                 i, i + 4 Staple 
               
               
                   
               
               
                 52 
                 S5-RLF-S5-SY 
                 7 
                 i, i + 4 Staple 
               
               
                   
               
               
                 53 
                 KQKRKFS-S5-FFK-S5-L 
                 13 
                 i, i + 4 Staple 
               
               
                   
               
               
                 54 
                 KQKRK-pff-S-S5-FFK-S5-L 
                 13 
                 i, i + 4 Staple 
               
               
                   
               
               
                 54 
                 KQKRK-pff-S-S5-FFK-S5-L 
                 13 
                 i, i + 4 Staple 
               
               
                   
               
               
                 55 
                 KF-S5-RLF-S5 
                 7 
                 i, i + 4 Staple 
               
               
                   
               
               
                 56 
                 S5-RLF-S5 
                 5 
                 i, i + 4 Staple 
               
               
                   
               
               
                 57 
                 RKF-S5-RLF-S5 
                 8 
                 i, i + 4 Staple 
               
               
                   
               
               
                 58 
                 KQKRKFS-S5-FFK-S5-LV 
                 13 
                 i, i + 4 Staple 
               
               
                   
               
               
                 59 
                 KQ-pff-RKKS-S5-FFK-S5-L 
                 13 
                 i, i + 4 Staple 
               
               
                   
               
               
                 60 
                 RK-pff-S5-RLF-S5 
                 8 
                 i, i + 4 Staple 
               
               
                   
               
               
                 61 
                 F-S5-RLF-S5 
                 6 
                 i, i + 4 Staple 
               
               
                   
               
               
                 62 
                 KTYRGAFQ-S5-LFQ-S5-VRE 
                 16 
                 i, i + 4 Staple 
               
               
                   
               
               
                 63 
                 STALR-S5-LIE-S5-LVNITQNQKAPL 
                 22 
                 i, i + 4 Staple 
               
               
                   
               
               
                 64 
                 STALRELI-S5-ELV-S5-ITQNQKAPL 
                 22 
                 i, i + 4 Staple 
               
               
                   
               
               
                 65 
                 STALRELIEEL-S5-NIT-S5-NQKAPL 
                 22 
                 i, i + 4 Staple 
               
               
                   
               
               
                 66 
                 NELK-S5-SFF-S5-LRDQIPELENNEKAP 
                 24 
                 i, i + 4 Staple 
               
               
                   
               
               
                 67 
                 LENRQ-S5-KLE-S5-ANRHLL 
                 16 
                 i, i + 4 Staple 
               
               
                   
               
               
                 68 
                 IL-S5-ASV-S5-YIRKLQREQ 
                 16 
                 i, i + 4 Staple 
               
               
                   
               
               
                 69 
                 FNI-S5-DRI-S5-ELGTLI 
                 14 
                 i, i + 4 Staple 
               
               
                   
               
               
                 70 
                 KN-S5-THQ-S5-DI 
                 9 
                 i, i + 4 Staple 
               
               
                   
               
               
                 71 
                 STALRELIEELV-S5-ITQ-S5-QKAPL 
                 21 
                 i, i + 4 Staple 
               
               
                   
               
               
                 72 
                 NELK-S5-SFF-S5-LRDQI 
                 14 
                 i, i + 4 Staple 
               
               
                   
               
               
                 73 
                 LENRQKKLE-S5-ANR-S5-LL 
                 16 
                 i, i + 4 Staple 
               
               
                   
               
               
                 74 
                 ILKAS-S5-DYI-S5-KLQREQ 
                 16 
                 i, i + 4 Staple 
               
               
                   
               
               
                 75 
                 DHIK-S5-SFH-S5-LRDSV 
                 14 
                 i, i + 4 Staple 
               
               
                   
               
               
                 76 
                 DHIKDSF-S5-SLR-S5-SV 
                 14 
                 i, i + 4 Staple 
               
               
                   
               
               
                 77 
                 S5-Y1Q-S5- 
                 38 
                 i, i + 4 Staple 
               
               
                   
                 NLRRKNHTHQQDIDDLLKRQNALLEQQVR 
                   
                   
               
               
                   
                 ALGG 
                   
                   
               
               
                   
               
               
                 78 
                 TYRGAAQ-S5-AAQ-S5-VREV 
                 16 
                 i, i + 4 Staple 
               
               
                   
               
               
                 79 
                 TY-S5-GAF-S5-NLFQSVREV 
                 16 
                 i, i + 4 Staple 
               
               
                   
               
               
                 80 
                 A-S5-SVF-S5-NYFHSVPYFEL 
                 17 
                 i, i + 4 Staple 
               
               
                   
               
               
                 81 
                 GAF-S5-NLF-S5-SV 
                 10 
                 i, i + 4 Staple 
               
               
                   
               
               
                 82 
                 S5-GAF-S5-NLF-R5-SV 
                 11 
                 i, i + 4 Staple 
               
               
                   
               
               
                 83 
                 SYRGAFQ-S5-LFQ-S5-VREV 
                 16 
                 i, i + 4 Staple 
               
               
                   
               
               
                 84 
                 SSVFY-S5-YFH-S5-VPYFEL 
                 16 
                 i, i + 4 Staple 
               
               
                   
               
               
                 85 
                 A-S5-TLF-S5-TFYLGGLLG 
                 15 
                 i, i + 4 Staple 
               
               
                   
               
               
                 86 
                 S5-GAF-S5-NLFQSV 
                 11 
                 i, i + 4 Staple 
               
               
                   
               
               
                 87 
                 A-S5-SSF-S5-DFFHTVPYNLL 
                 17 
                 i, i + 4 Staple 
               
               
                   
               
               
                 88 
                 ERLRRRI-S5-LCR-S5-HHST 
                 16 
                 i, i + 4 Staple 
               
               
                   
               
               
                 89 
                 ERLRRRI-S5-NLCR-S5-HHST 
                 17 
                 i, i + 4 Staple 
               
               
                   
               
               
                 90 
                 ERLRRRL-S5-LCR-S5-HHST 
                 16 
                 i, i + 4 Staple 
               
               
                   
               
               
                 91 
                 ERLRRRF-S5-LCR-S5-HHST 
                 16 
                 i, i + 4 Staple 
               
               
                   
               
               
                 92 
                 ERFRRRI-S5-LCR-S5-HHST 
                 16 
                 i, i + 4 Staple 
               
               
                   
               
               
                 93 
                 ERLARRI-S5-LCR-S5-HHST 
                 16 
                 i, i + 4 Staple 
               
               
                   
               
               
                 94 
                 ENPESILD-S5-HVQ-S5-VM 
                 15 
                 i, i + 4 Staple 
               
               
                   
               
               
                 95 
                 PE-S5-ILD-S5-HVQRVM 
                 13 
                 i, i + 4 Staple 
               
               
                   
               
               
                 96 
                 ERLRRRI-S5-FCR-S5-HHST 
                 16 
                 i, i + 4 Staple 
               
               
                   
               
               
                 97 
                 ERLRRRNL-S5-LCR-S5-HHST 
                 17 
                 i, i + 4 Staple 
               
               
                   
               
               
                 98 
                 ERNLRRRI-S5-LCR-S5-HHST 
                 17 
                 i, i + 4 Staple 
               
               
                   
               
               
                 99 
                 ERWRRRI-55-LCR-55-HHST 
                 16 
                 i, i + 4 Staple 
               
               
                   
               
               
                 100 
                 RELRREI-S5-LCR-S5-HHST 
                 16 
                 i, i + 4 Staple 
               
               
                   
               
               
                 101 
                 ENPE-S5-ILD-S5-HVQRVM 
                 15 
                 i, i + 4 Staple 
               
               
                   
               
               
                 102 
                 NPE-S5-ILD-S5-HVQRVM 
                 14 
                 i, i + 4 Staple 
               
               
                   
               
               
                 103 
                 WPE-S5-ILD-S5-HVQRVM 
                 14 
                 i, i + 4 Staple 
               
               
                   
               
               
                 104 
                 PE-S5-ILD-S5-HVRRVMR 
                 14 
                 i, i + 4 Staple 
               
               
                   
               
               
                 105 
                 RPE-S5-ILD-S5-HVRRVMR 
                 15 
                 i, i + 4 Staple 
               
               
                   
               
               
                 106 
                 TRQA-R8-RNRRRR-S5-RR 
                 14 
                 i, i + 7 Staple 
               
               
                   
               
               
                 107 
                 RRGSRPSGA-R8-RRRRRA-S5 
                 17 
                 i, i + 7 Staple 
               
               
                   
               
               
                 108 
                 RRGSRPSGA-R8-RRRRRA-S5-AA 
                 19 
                 i, i + 7 Staple 
               
               
                   
               
               
                 109 
                 TRQARRN-R8-RRRWRE-S5-QR 
                 17 
                 i, i + 7 Staple 
               
               
                   
               
               
                 110 
                 RRRR-R5-RRRWRR-S8 
                 12 
                 i, i + 7 Staple 
               
               
                   
               
               
                 111 
                 KPE-S5-ILD-S5-HVQRVM 
                 14 
                 i, i + 7 Staple 
               
               
                   
               
               
                 112 
                 WPE-S5-ILD-S5-HVRRVMR 
                 15 
                 i, i + 7 Staple 
               
               
                   
               
               
                 113 
                 RRRR-R8-RQRRRR-S5-RR 
                 14 
                 i, i + 7 Staple 
               
               
                   
               
               
                 114 
                 RRGSRPSGA-R8-RRRRRR-S5 
                 17 
                 i, i + 7 Staple 
               
               
                   
               
               
                 115 
                 R8-RRQRRR-S5-RQRRRRR 
                 15 
                 i, i + 7 Staple 
               
               
                   
               
               
                 109 
                 TRQARRN-R5-RRRWRE-S8-QR 
                 17 
                 i, i + 7 Staple 
               
               
                   
               
               
                 116 
                 RRRR-R5-RRRRRR-S8 
                 12 
                 i, i + 7 Staple 
               
               
                   
               
               
                 117 
                 YGRK-R5-RRQRRR-S8 
                 12 
                 i, i + 7 Staple 
               
               
                   
               
               
                 118 
                 S-R8-ELVAEA-S5-NLCTLLENAIQDTVREQ 
                 25 
                 i, i + 7 Staple 
               
               
                   
               
               
                 119 
                 SEELVAEAH-R8-LCTLLE-S5-AIQDTVREQ 
                 26 
                 i, i + 7 Staple 
               
               
                   
               
               
                 120 
                 SEELVAEAHNLCT-R8-LENAIQ-S5-TVREQ 
                 26 
                 i, i + 7 Staple 
               
               
                   
               
               
                 121 
                 RQIKIW-R5-QNRRMK-S8-KK 
                 16 
                 i, i + 7 Staple 
               
               
                   
               
               
                 110 
                 RRRR-R5-RRRWRR-S8 
                 12 
                 i, i + 7 Staple 
               
               
                   
               
               
                 118 
                 S-R8-ELVAEA-S5-NLCTLLENAIQDTVREQ 
                 26 
                 i, i + 7 Staple 
               
               
                   
               
               
                 122 
                 SE-R8-LVAEAH-S5-LCTLLENAIQDTVREQ 
                 26 
                 i, i + 7 Staple 
               
               
                   
               
               
                 123 
                 SEELVAEAHNLC-R8-LLENAI-S5-DTVREQ 
                 26 
                 i, i + 7 Staple 
               
               
                   
               
               
                 124 
                 SEELVAEAHNLCTLLE-R8-AIQDTV-S5-EQ 
                 26 
                 i, i + 7 Staple 
               
               
                   
               
               
                 125 
                 LSQETF-R8-DLWKLL-S5-EN 
                 16 
                 i, i + 7 Staple 
               
               
                   
               
               
                 126 
                 ILR-R8-AVSHMK-S5-LRGT 
                 15 
                 i, i + 7 Staple 
               
               
                   
               
               
                 126 
                 ILR-R8-AVSHMK-S5-LRGT 
                 15 
                 i, i + 7 Staple 
               
               
                   
               
               
                 127 
                 NEL-R8-RSFRSL-S5-DSI 
                 14 
                 i, i + 7 Staple 
               
               
                   
               
               
                 128 
                 NEL-R8-RSFRAL-S5-DQI 
                 14 
                 i, i + 7 Staple 
               
               
                   
               
               
                 129 
                 NEL-R8-RSFFAL-S5-DSI 
                 14 
                 i, i + 7 Staple 
               
               
                   
               
               
                 130 
                 NEL-R8-RSFFAL-S5-DQI 
                 14 
                 i, i + 7 Staple 
               
               
                   
               
               
                 131 
                 IL-R8-MA-VSHM-S5-SLRGT 
                 15 
                 i, i + 7 Staple 
               
               
                   
               
               
                 132 
                 NEL-R8-RSFRAL-S5-DSI 
                 14 
                 i, i + 7 Staple 
               
               
                   
               
               
                 133 
                 NEL-R8-RSFFSL-S5-DQI 
                 14 
                 i, i + 7 Staple 
               
               
                   
               
               
                 134 
                 WNEL-R8-RSFRSL-S5-DQI 
                 15 
                 i, i + 7 Staple 
               
               
                   
               
               
                 135 
                 NQR-R8-LSFFAL-S5-DQI 
                 14 
                 i, i + 7 Staple 
               
               
                   
               
               
                 14 
                 NQL-R8-RSFFAL-S5-DQI 
                 14 
                 i, i + 7 Staple 
               
               
                   
               
               
                 135 
                 NQR-R8-LSFFAL-S5-DQI 
                 14 
                 i, i + 7 Staple 
               
               
                   
               
               
                 136 
                 NQL-R8-LSFFAR-S5-DQI 
                 14 
                 i, i + 7 Staple 
               
               
                   
               
               
                 137 
                 NKL-R8-RSFFAL-S5-DQI 
                 14 
                 i, i + 7 Staple 
               
               
                   
               
               
                 130 
                 NEL-R8-RSFFAL-S5-DQI 
                 14 
                 i, i + 7 Staple 
               
               
                   
               
               
                 138 
                 NELK-R8-SFFALR-S5-QIPELENNEKAP 
                 24 
                 i, i + 7 Staple 
               
               
                   
               
               
                 139 
                 AHL-R8-LCLEKL-S5-GLV 
                 14 
                 i, i + 7 Staple 
               
               
                   
               
               
                 14 
                 NQL-R8-RSFFAL-S5-DQI (D-amino acids) 
                 14 
                 i, i + 7 Staple 
               
               
                   
               
               
                 191 
                 IQD-S5-LAFFSR-R8-LQN (D-amino acids) 
                 14 
                 i, i + 7 Staple 
               
               
                   
               
               
                 140 
                 NKL-R8-RS-FKAL-S5-KQI 
                 14 
                 i, i + 7 Staple 
               
               
                   
               
               
                 141 
                 NELK-R8-S-FFALR-S5-QI 
                 14 
                 i, i + 7 Staple 
               
               
                   
               
               
                 142 
                 NQL-R8-RS-FFAL-S5-DQIPELENNEKAP 
                 24 
                 i, i + 7 Staple 
               
               
                   
               
               
                 14 
                 NQL-R8-RSFFAL-S5-DQI 
                 14 
                 i, i + 7 Staple 
               
               
                   
               
               
                 192 
                 AHL-R8-LCLEKL-S5-GLV-(K-(PEG)1- 
                 15 
                 i, i + 7 Staple 
               
               
                   
               
               
                 143 
                 KV-R8-ILKKAT-S5-YILS 
                 14 
                 i, i + 7 Staple 
               
               
                   
               
               
                 144 
                 R8-KRRAHA-S5-AERARR 
                 14 
                 i, i + 7 Staple 
               
               
                   
               
               
                 191 
                 IQD-S5-LAFFSR-R8-LQN (D-amino acids) 
                 14 
                 i, i + 7 Staple 
               
               
                   
               
               
                 14 
                 NQL-R8-RSFFAL-S5-DQI (D-amino acids) 
                 14 
                 i, i + 7 Staple 
               
               
                   
               
               
                 14 
                 NQL-R8-RS-FFAL-S5-DQI 
                 14 
                 i, i + 7 Staple 
               
               
                   
               
               
                 14 
                 NQL-R8-RSFFAL-S5-DQI 
                 14 
                 i, i + 7 Staple 
               
               
                   
               
               
                 145 
                 EENAKRR-R8-HNALER-S5-RR 
                 17 
                 i, i + 7 Staple 
               
               
                   
               
               
                 193 
                 NQL-R8-FSRFAL-S5-DQI (D-amino acids) 
                 14 
                 i, i + 7 Staple 
               
               
                   
               
               
                 146 
                 NQL-R8-LS-S5-DQI 
                 10 
                 i, i + 7 Staple 
               
               
                   
               
               
                 147 
                 NQL-R8-FS-S5-DQI 
                 10 
                 i, i + 7 Staple 
               
               
                   
               
               
                 148 
                 TILKASVDYIRKL-R8-REQQRA-S5-EL 
                 23 
                 i, i + 7 Staple 
               
               
                   
               
               
                 149 
                 FNI-R8-DRI-S5-TLI 
                 11 
                 i, i + 7 Staple 
               
               
                   
               
               
                 150 
                 RNI-R8-DRI-S5-TRI 
                 11 
                 i, i + 7 Staple 
               
               
                   
               
               
                 151 
                 KATEYIQYNLRRKN-R8-THQQDI-S5-DL 
                 24 
                 i, i + 7 Staple 
               
               
                   
               
               
                 152 
                 NEL-R8-RSFFAL-S5-DQIDQIPAAKRVKLD 
                 26 
                 i, i + 7 Staple 
               
               
                   
               
               
                 14 
                 NQL-R8-RSFFAL-S5-DQI 
                 14 
                 i, i + 7 Staple 
               
               
                   
               
               
                 153 
                 RNI-R8-DRIKEL-S5-TLI 
                 14 
                 i, i + 7 Staple 
               
               
                   
               
               
                 154 
                 FNIN-R8-RIKELG-S5-LI 
                 14 
                 i, i + 7 Staple 
               
               
                   
               
               
                 155 
                 FNI-R8-DRIKEL-S5-TRI 
                 14 
                 i, i + 7 Staple 
               
               
                   
               
               
                 156 
                 NQL-R8-RSFRAL-S5-DQI 
                 15 
                 i, i + 7 Staple 
               
               
                   
               
               
                 157 
                 NEL-R8-RSFFAL-S5-DQIDQIPKKKRKV 
                 24 
                 i, i + 7 Staple 
               
               
                   
               
               
                 158 
                 ENPE-R8-ILDEHV-S5-RVM 
                 15 
                 i, i + 7 Staple 
               
               
                   
               
               
                 164 
                 S8-RQARRN-B5-RRRWRE-S8-QR 
                 16 
                 i, i + 4, i + 11 
               
               
                   
                   
                   
                 Stitch Reduced 
               
               
                   
               
               
                 163 
                 TRQ-S5-RRN-B5-RRRWRE-S8-QR 
                 17 
                 i, i + 4, i + 11 Stitch 
               
               
                   
               
               
                 159 
                 TRQ-S5-RRA-B5-RRRWRE-S8-QR 
                 17 
                 i, i + 4, i + 11 Stitch 
               
               
                   
               
               
                 160 
                 S5-RRN-B5-RRRWRE-S8 
                 12 
                 i, i + 4, i + 11 Stitch 
               
               
                   
               
               
                 161 
                 EY1Q-R5-NLRRKNH-S8-HQQDIDDLKRQNALLEQQVRALGG 
                 37 
                 i, i + 4, i + 11 Stitch 
               
               
                   
               
               
                 165 
                 S8-RQARRQ-B5-RRRWRE-S8-QR 
                 17 
                 i, i + 4, i + 11 
               
               
                   
                   
                   
                 Stitch Reduced 
               
               
                   
               
               
                 162 
                 TRQ-S5-Q-B5-RRRWRE-S8-QR 
                 15 
                 i, i + 4, i + 11 Stitch 
               
               
                   
               
               
                 163 
                 TRQ-S5-RRN-B5-RRRWRE-S8-QR 
                 17 
                 i, i + 4, i + 11 
               
               
                   
                   
                   
                 Stitch Reduced 
               
               
                   
               
               
                 164 
                 R8-RQARRN-B5-RRRWRE-S8-QR 
                 17 
                 i, i + 4, i + 11 
               
               
                   
                   
                   
                 Stitch Reduced 
               
               
                   
               
               
                 165 
                 R8-RQARRQ-B5-RRRWRE-S8-QR 
                 17 
                 i, i + 4, i + 11 Stitch 
               
               
                   
               
               
                 166 
                 S5-RRN-B5-RRRWRR-S8 
                 12 
                 i, i + 4, i + 11 Stitch 
               
               
                   
               
               
                 167 
                 RRA-B5-RRRWRR-S8 
                 11 
                 i, i + 4, i + 11 Stitch 
               
               
                   
               
               
                 169 
                 S5-RRR-B5-RRRRRR-S8 
                 12 
                 i, i + 4, i + 11 Stitch 
               
               
                   
               
               
                 168 
                 S5-KIW-B5-QNRRNLK-S8 
                 13 
                 i, i + 4, i + 11 Stitch 
               
               
                   
               
               
                 169 
                 S5-RRR-B5-RRRRRR-S8 
                 12 
                 i, i + 4, i + 11 Stitch 
               
               
                   
               
               
                 170 
                 S5-GRK-B5-RRQRRR-S8 
                 12 
                 i, i + 4, i + 11 Stitch 
               
               
                   
               
               
                 171 
                 S5-RRQ-B5-RRRWRR-S8 
                 12 
                 i, i + 4, i + 11 Stitch 
               
               
                   
               
               
                 174 
                 S5-RRR-B5-RRRWRR-S8 
                 12 
                 i, i + 4, i + 11 Stitch 
               
               
                   
               
               
                 172 
                 RQ-S5-KIW-B5-QNRRMK-S8-KK 
                 16 
                 i, i + 4, i + 11 Stitch 
               
               
                   
               
               
                 173 
                 S5-KIW-B5-QNRRAK-S8 
                 12 
                 i, i + 4, i + 11 Stitch 
               
               
                   
               
               
                 174 
                 S5-RRR-B5-RRRWRR-S8 
                 12 
                 i, i + 4, i + 11 Stitch 
               
               
                   
               
               
                 175 
                 L-S5-ILQ-B5-AVQVIL-S8-LEQQVRER 
                 21 
                 i, i + 4, i + 11 Stitch 
               
               
                   
               
               
                 176 
                 LLILQQAV-S5-VIL-B5-LEQQVR-S8-R 
                 21 
                 i, i + 4, i + 11 Stitch 
               
               
                   
               
               
                 182 
                 S5-DFS-B5-YWK-R5-L 
                 10 
                 i, i + 4, i + 11 Stitch 
               
               
                   
               
               
                 177 
                 LS-S5-ETF-B8-DLWKLL-S8-EN 
                 16 
                 i, i + 4, i + 11 Stitch 
               
               
                   
               
               
                 178 
                 LSQ-S5-TFS-B8-LWKLLA-S8-N 
                 16 
                 i, i + 4, i + 11 Stitch 
               
               
                   
               
               
                 179 
                 L-S5-ILQ-B5-AVQ-R5-ILGLEQQVRER 
                 21 
                 i, i + 4, i + 11 Stitch 
               
               
                   
               
               
                 180 
                 LLILQQAV-S5-VIL-B5-LEQ-R5-VRER 
                 21 
                 i, i + 4, i + 11 Stitch 
               
               
                   
               
               
                 181 
                 LLIL-S5-QAV-B5-VIL-R5-LEQQVRER 
                 21 
                 i, i + 4, i + 11 Stitch 
               
               
                   
               
               
                 182 
                 R5-DFS-B5-YVVK-S5-L 
                 10 
                 i, i + 4, i + 11 Stitch 
               
               
                   
               
               
                 183 
                 LS-S5-ETA-B8-DLWKLL-S8-EN 
                 16 
                 i, i + 4, i + 11 Stitch 
               
               
                   
               
               
                 184 
                 EDIIRNIA-S5-HLA-B5-VGDWNLD-S8-SI 
                 23 
                 i, i + 4, i + 11 Stitch 
               
               
                   
               
               
                 185 
                 NIA-S5-HLA-B5-VGDWNLD-S8-SI (isomer 2) 
                 18 
                 i, i + 4, i + 11 Stitch 
               
               
                   
               
               
                 186 
                 S5-HLA-B5-VGDWNLD-S8 (isomer 1) 
                 13 
                 i, i + 4, i + 11 Stitch 
               
               
                   
               
               
                 187 
                 NVKRR-R8-HNVLER-S5-RRNEL-R8-RSFFAL-S5-DQI 
                 29 
                 i, i + 4, i + 11 Stitch 
               
               
                   
               
               
                 188 
                 S5-Y1Q-B5-NLRRKNH-S8- 
                 38 
                 i, i + 4, i + 11 Stitch 
               
               
                   
                 HQQDIDDLLKRQNALLEQQVRALGG 
                   
                   
               
               
                   
               
               
                 185 
                 NIA-S5-HLA-B5-VGDWNLD-S8-SI 
                 18 
                 i, i + 4, i + 11 Stitch 
               
               
                   
               
               
                 189 
                 NIA-S5-HLA-B5-VGDWNLD-S8 
                 16 
                 i, i + 4, i + 11 Stitch 
               
               
                   
               
               
                 186 
                 S5-HLA-B5-VGDWNLD-S8 
                 13 
                 i, i + 4, i + 11 Stitch 
               
               
                   
               
               
                 190 
                 EYIQYNLR-S5-KNH-B5-HQQDID-S8- 
                 37 
                 i, i + 4, i + 11 Stitch 
               
               
                   
                 LKRQNALLEQQVRALGG 
               
               
                   
               
               
                 S5 = a-methyl, a-alkenylglycine with 5 carbon chain 
               
               
                 S8 = a-methyl, a-alkenylglycine with 8 carbon chain 
               
               
                 B5 = a-methyl, a-alkenylglycine with two 5 carbon chain 
               
            
           
         
       
     
     Alternative CPPs and their method of manufacture are disclosed in Chu et al, 2014 and associated supplementary information, and are incorporated by reference 21 . 
     The exemplified stabilized peptide comprises two or more olefin bearing side chains that are covalently formed, typically by means of a ring-closing metathesis. 
     The stabilized conformation typically comprises at least one alpha helix. It may however, in the alternative, comprise at least one turn (for example, but not limited to, α, β, γ, δ or π), several turns to form a beta sheet, or a combination of one or more of: an alpha helix, turn, or beta sheet. 
     The formal charge of a CPP is calculated at physiological pH (about 7.5) and is based on the pKa of amino acid R groups. These values (pK x ) are represented in Table 3. 
     
       
         
           
               
               
               
               
               
               
               
               
               
               
               
             
               
                 TABLE 3 
               
               
                   
               
               
                   
                   
                   
                   
                   
                   
                 Residue 
                   
                   
                   
                   
               
               
                   
                 3-Letter 
                 1-Letter 
                 Molecular 
                 Molecular 
                 Residue 
                 Weight 
                   
                   
                   
                   
               
               
                 Name 
                 Symbol 
                 Symbol 
                 Weight 
                 Formula 
                 Formula 
                 (—H 2 O) 
                 pKa 1   
                 pKb 2   
                 pKx 3   
                 pI 4   
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Alganine 
                 Ala 
                 A 
                 89.10 
                 C 3 H 7 NO 2   
                 C 3 H 5 NO 
                 71.08 
                 2.34 
                 9.69 
                 — 
                 6.00 
               
               
                 Arginine 
                 Arg 
                 R 
                 174.20 
                 C 6 H 14 N 4 O 2   
                 C 6 H 12 N 4 O 
                 156.19 
                 2.17 
                 9.04 
                 12.48 
                 10.76 
               
               
                 Asparagine 
                 Asn 
                 N 
                 132.12 
                 C 4 H 8 N 2 O 3   
                 C 4 H 6 N 2 O 2   
                 114.11 
                 2.02 
                 8.80 
                 — 
                 5.41 
               
               
                 Aspartic Acid 
                 Asp 
                 D 
                 133.11 
                 C 4 H 7 NO 4   
                 C 4 H 5 NO 3   
                 115.09 
                 1.88 
                 9.60 
                 3.65 
                 2.77 
               
               
                 Cysteine 
                 Cys 
                 C 
                 121.16 
                 C 3 H 7 NO 2 S 
                 C 3 H 5 NOS 
                 103.15 
                 1.96 
                 10.28 
                 8.18 
                 5.07 
               
               
                 Glutamic acid 
                 Glu 
                 E 
                 147.13 
                 C 5 H 9 NO 4   
                 C 5 H 7 NO 3   
                 129.12 
                 2.19 
                 9.67 
                 4.25 
                 3.22 
               
               
                 Glutamine 
                 Gln 
                 Q 
                 146.15 
                 C 5 H 10 N 2 O 3   
                 C 5 H 8 N 2 O 2   
                 128.13 
                 2.17 
                 9.13 
                 — 
                 5.65 
               
               
                 Glycine 
                 Gly 
                 G 
                 75.07 
                 C 2 H 5 NO 2   
                 C 2 H 3 NO 
                 57.05 
                 2.34 
                 9.60 
                 — 
                 5.97 
               
               
                 Histidine 
                 His 
                 H 
                 155.16 
                 C 6 H 9 N 3 O 2   
                 C 6 H 7 N 3 O 
                 137.14 
                 1.82 
                 9.17 
                 6.00 
                 7.59 
               
               
                 Hydroxyproline 
                 Hyp 
                 O 
                 131.13 
                 C 5 H 9 NO 3   
                 C 5 H 7 NO 2   
                 113.11 
                 1.82 
                 9.65 
                 — 
                 — 
               
               
                 Isoleucine 
                 Ile 
                 I 
                 131.18 
                 C 6 H 13 NO 2   
                 C 6 H 11 NO 
                 113.16 
                 2.36 
                 9.60 
                 — 
                 6.02 
               
               
                 Leucine 
                 Leu 
                 L 
                 131.18 
                 C 6 H 13 NO 2   
                 C 6 H 11 NO 
                 113.16 
                 2.36 
                 9.60 
                 — 
                 5.98 
               
               
                 Lysine 
                 Lys 
                 K 
                 146.19 
                 C 6 H 14 N 2 O 2   
                 C 6 H 12 N 2 O 
                 128.18 
                 2.18 
                 8.95 
                 10.53 
                 9.74 
               
               
                 Methionine 
                 Met 
                 M 
                 149.21 
                 C 5 H 11 NO 2 S 
                 C 5 H 11 NOS 
                 131.20 
                 2.28 
                 9.21 
                 — 
                 5.74 
               
               
                 Phenylalanine 
                 Phe 
                 F 
                 165.19 
                 C 9 H 11 NO 2   
                 C 9 H 9 NO 
                 147.18 
                 1.83 
                 9.13 
                 — 
                 5.48 
               
               
                 Proline 
                 Pro 
                 P 
                 115.13 
                 C 5 H 9 NO 2   
                 C 5 H 7 NO 
                 97.12 
                 1.99 
                 10.60 
                 — 
                 6.30 
               
               
                 Pyroglutamatic 
                 Glp 
                 U 
                 139.11 
                 C 5 H 7 NO 3   
                 C 5 H 5 NO 2   
                 121.09 
                 — 
                 — 
                 — 
                 5.68 
               
               
                 Serine 
                 Ser 
                 S 
                 105.09 
                 C 3 H 7 NO 3   
                 C 3 H 5 NO 2   
                 87.08 
                 2.21 
                 9.15 
                 — 
                 5.68 
               
               
                 Threonine 
                 Thr 
                 T 
                 119.12 
                 C 4 H 9 NO 3   
                 C 4 H 7 NO 2   
                 101.11 
                 2.09 
                 9.10 
                 — 
                 5.60 
               
               
                 Tryptophan 
                 Trp 
                 W 
                 204.23 
                 C 11 H 12 N 2 O 2   
                 C 11 H 10 N 2 O 
                 186.22 
                 2.83 
                 9.39 
                 — 
                 5.89 
               
               
                 Tyrosine 
                 Tyr 
                 Y 
                 181.19 
                 C 9 H 11 NO 3   
                 C 9 H 9 NO 2   
                 163.18 
                 2.20 
                 9.11 
                 10.07 
                 5.66 
               
               
                 Valine 
                 Val 
                 V 
                 117.15 
                 C 5 H 11 NO 2   
                 C 5 H 9 NO 
                 99.13 
                 2.32 
                 9.62 
                 — 
                 5.96 
               
               
                   
               
               
                   1 pK a  is the negative of the logarithm of the dissociated constant for the-COOH group 
               
               
                   2 pK b  is the negative of the logarithm of the dissociated constant for the-NH 3   +  group 
               
               
                   3 pK x  is the negative of the logarithm of the dissociated constant for any other group in the molecule 
               
               
                   4 pI is the pH at the isoelectric point 
               
               
                 References: D. R. Lide, Handbook of Chemistry and Physics, 72nd Edition, CRC Press, Boca Raton, FL, 1991. 
               
            
           
         
       
     
     CPPs typically used to date harbour many positively charged residues. Reducing the amount of positively charged residues within the amino acid sequence, whilst retaining the ability to cross a biological membrane, will be more clinically relevant. 
     Accordingly, it is possible to reduce the charge on the peptide sequences illustrated in Table 2. 
     The preferred BAC is an oligonucleotide (ON), more preferably still an anti-sense oligonucleotide (AON). Different anti-sense oligonucleotide chemistries are illustrated in Table 4, with the use of low charge or neutral charged chemistries, such as, phosphorodiamidate morpholino oligonucleotides (PMOs) being preferred. 
     
       
         
           
               
               
               
             
               
                 TABLE 4 
               
               
                   
               
             
            
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
            
           
         
       
     
     The BAC may target and alter the expression of an endogenous or exogenous gene. Endogenous gene targets include but are not limited to genes associated with neuromuscular disease, metabolic disease, cancer, age-related degenerative diseases, and exogenous gene targets include those of an acquired disease e.g. viral infections. 
     Whilst the BAC may be linked to the CPP directly the Applicant has found the use of a BFL desirable. Exemplary, non-limiting BFL chemistries are illustrated in Table 5. 
     
       
         
           
               
               
               
               
               
             
               
                 TABLE 5 
               
               
                   
               
               
                   
                   
                 Linker  
                   
                   
               
               
                   
                   
                 acronym 
                   
                   
               
               
                 Entry 
                 Linker (L) 
                 is present 
                 Z 
                 Y 3   
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 1 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 SMCC 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 2 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 AMAS 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 3 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 BMPS 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 4 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 GMPS 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 5 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 DMVS 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 6 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 EMCS 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 7 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 LC- SMCC 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 8 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 SM(PEG) n   
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 9 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 DSG 
                 Not present 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 10 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 DSCDS 
                 Not present 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 11 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 HNA 
                 Not present 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
            
           
         
       
     
     By way of a footnote to Table 5, the following should be noted: 
       FIG.  5 A  highlights general structure of a DCCPM where the following are preferred, but not limited to the following defined atoms or groups. 
     In a preferred embodiment illustrated in  FIG.  5 C , where Y1=Nitrogen, Y2=Hydrogen, Y3=spacer such as (PEG)n n=5, but not limited to those identified in Table 5, Z=a sulfur containing moiety e.g. Cysteine and L=BFL such as SMCC 
     Other embodiments may utilize variations over the structure shown in  FIG.  5 A . For example if another embodiment does not require a thiol for conjugation of the BFL to the CPA as illustrated in  FIG.  5 D , then Z=Y3 where Y3 is a spacer in Table 5. For a BFL that does not require a sulphur for conjugation of the BAC and CPA e.g. not limited to entries 9-11 in Table 5 Z=a covalent bond between L and Y3 
     Other embodiments may not require the use of a spacer, a BFL and as such a thiol group for the formation of a DCCPM depicted in  FIG.  7    then the following apply. If no spacer is utilized then Y3 can represent a covalent bond between Y1 and the BAC in which case Z and L=Y1 where Y1 is a N terminus of the CPA. 
     These chemistries may be further expanded and Table 6 exemplifies modifications to amino acids via which functional groups can be introduced to provide desirable properties to the DCCPM. These will include, but are not limited to, an acetyl, a cholesterol, a fatty acid, a polyethylene glycol, a polysaccharide, an aminoglycan, a glycolipid, a polyphenol, a nuclear localising signal, a nuclear export signal, an antibody, and a targeting molecule. 
     
       
         
           
               
               
               
             
               
                 TABLE 6 
               
               
                   
               
               
                 Functional  
                 Reacting Functional  
                 Resulting  
               
               
                 group (X) 
                 Group (X 1 ) 
                 Functional Group 
               
               
                   
               
             
            
               
                 Aldehyde 
                 Hydrazine 
                 Hydrazone 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 Thiol 
                 Maleimides 
                 Thioether 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 Thiol 
                 Thiol 
                 Disulfide 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 Alkyne 
                 Azide 
                 Triazole 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 Amine 
                 Carboxylic acid 
                 Amide 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 Diazirine 
                   
                 Various 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 Any 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 Olefin 
                 Olefin 
                 Olefin 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 Aryl or Olefin 
                 Halide 
                   
               
               
                 R—M  
                 R 2 —X  
                 R—R 2   
               
               
                   
               
            
           
         
       
     
     A preferred linker chemistry utilises an amine to sulphydryl cross linker containing N-hydroxysuccinimide esters and malemide reactive groups separated by a cyclohexane spacer namely succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) to form a covalent bond between the BFL and the CPP. 
     A schematic intermediate compound using SMCC as the BFL and the resultant DCCPM is depicted in  FIG.  5     b.    
     In a particularly preferred embodiment the linker may incorporate polyethylene glycol in single or multiple units (PEG) n , where n=1 to 10 PEG molecules. 
     Hereafter, where the CPP comprises the sequence RKF-S5-RLF-S5 (SEQ ID NO: 57) and the BFL is a PEGylated SMCC, the resultant compound is termed CP8M. 
     Where the CPP comprises the sequence RKF-S5-RLF-S5 (SEQ ID NO: 57) and the BFL is a PEGylated hydrazynal nicotinic acid (HNA), the resultant compound is termed HP8M. 
     Where the CPP comprises the sequence RKF-S5-RLF-S5 (SEQ ID NO: 57) and the BFL is SMCC, the resultant compound is termed CBM. 
     Thus a CPA, such as Compound III ( FIG.  5   b   ) may be covalently linked to a BFL, if required, preferentially incorporating (PEG) n  where n=1-10. 
     Covalent linkage to the CPP may be via, for example, but not limited to, a β-ala or any other suitable moiety. 
     In the preferred embodiment, the (PEG) n  is linked using a sulphur containing molecule e.g. cysteine, to enable covalent coupling as a PEGylated (SMCC). This in turn is covalently linked to a functional group on the BAC, in the preferred embodiment a primary amine, (Compound I), thus generating a DCCPM (Compound V). 
     According to a second aspect of the invention there is provided a method for facilitating the uptake of a biologically active compound (BAC) into a cell by the conjugation of the biologically active compound, directly or via a bi-functional linker (BFL), to a cell penetrating agent (CPA) which is a stabilized peptide which has a conformation imposed upon it by stapling to form a stapled peptide (StaP) or stitching to form a stitched peptide (StiP), to form a drug carrying cell penetrating molecule (DCCPM) and presenting said DCCPM to said cell in a suitable vehicle. 
     Where HNA has been incorporated into the terminal end of the CPP, to form a DCCPM in which the BAC is an ON, the ON has been modified to incorporate 4 formyl benzioic acid to facilitate covalent conjugation. 
     According to a third aspect of the present invention there is provided a DCCPM of the first aspect of the invention for use in the treatment of a disease requiring alteration of the expression of an endogenous or exogenous gene. 
     The DCCPM may be used in the treatment of a, for example, neuromuscular disease, metabolic disease, cancer, age-related degenerative disease or to treat an acquired viral infection. 
     In one embodiment the DCCPM is used in the treatment of a muscular dystrophy e.g. Duchenne muscular dystrophy (DMD) although the skilled person will readily appreciate that the invention can be used to target a wide range of genes. 
     In the case of DMD the DCCPM may comprise an AON targeting exon 51 of the dystrophin gene. 
     In accordance with a fourth aspect of the present invention there is provided a method of improving the bioavailability of a drug or BAC comprising linking the drug or BAC to a CPP which is a stabilized peptide which has a conformation imposed upon it by stapling to form a stapled peptide (StaP) or stitching to form a stitched peptide (StiP). 
     In accordance with a fifth aspect of the present invention there is provided a method of introducing a drug or BAC to a site which is refractory to a drug or BAC in its native state comprising linking the drug or BAC to a CPP which is a stabilized peptide which has a conformation imposed upon it by stapling to form a stapled peptide (StaP) or stitching to form a stitched peptide (StiP) and administering it to a subject. 
     The DCCPMs of the invention can be used to administer the drug or BAC to a target tissue, such as, for example the heart, brain or muscle. 
     In accordance with a sixth aspect of the present invention there is provided a method of treating a subject to alter the expression of an endogenous or exogenous gene comprising administering a DCCPM of the invention to a subject. 
     In accordance with a seventh aspect of the present invention there is provided a composition comprising a DCCPM of the invention and one or more pharmaceutically acceptable excipients enabling the composition to be administered orally, parenterally, intravenously or topically. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       Embodiments of the invention are further described hereinafter with reference to the accompanying Drawings, in which: 
         FIG.  1   a - 1   c    show a general schematic of a CPP which it has been stabilized by means of the incorporation of olefin-bearing α, α-di-substituted amino acids that permit a further chemical modification such that a cross link can be formed. Cross linking of two such non-natural amino acids is termed stapling; cross linking involving more than two non-natural amino acids is termed stitching. This schematic can be referenced against Tables 1 and 2 of this application. 
         FIG.  1   a    is an example of a non-cyclised and an i,i+4 ring closing metathesis (RCM) using Grubb&#39;s Gen 1 catalyst to form an StaP CPA; 
         FIG.  1   b    is an example of a reduced RCM StaP using conventional reduction chemistry; 
         FIG.  1   c    is a schematic showing a selection of different StaP or StiP RCM configurations and their corresponding starting positions; 
         FIG.  2    exemplifies the structures of charge variants: 3+ (CP8M-3), 2+ (CP8M-2), 1+(CP8M-1) and 0+ (CP8M-0) illustrating relative positions of charge on the StaP, however the positions and charge can be varied in any permutation or combination; 
         FIG.  3    exemplifies the structure of an amino acid which can be incorporated into StaP or StiPs with varying functional groups as defined in Table 6. The functional groups can then be used for bio-conjugation; 
         FIG.  4   a    is a representation of a RCM reaction to form a StaP; 
         FIG.  4   b    shows the resulting CD spectrum of the StaP and non-cyclised stating peptide; 
         FIG.  4   c    is a COSY NMR spectra of CP8M; 
         FIG.  4   d    is a NOESY NMR Spectra of CP8M; 
         FIG.  4   e    is a TOCSY NMR spectra of CP8M; 
       The Spectra were collected at room temperature in H 2 O spiked with 10% D 2 O and 10 mM sodium acetate; 
         FIG.  5   a    is a schematic diagram of StaP DCCPM depicting variants of linkers and spacers as defined in Table 5; 
         FIG.  5   b    is a schematic diagram of DCCPM depicting the synthetic steps for the conjugation of a StaP to a PMO. The PMO is modified to yield a 5′ amine group (compound I); the heterobifunctional protein crosslinker Succinimidyl-4-[N-maleimidomethyl] cyclohexane-1-carboxylate (SMCC; compound II) is attached to 5′ amine group to yield compound IV; an i,i+4 stapled peptide (compound III) is conjugated to generate the final DCCPM (compound V); 
         FIG.  5   c    is a DCCPM of CP8M conjugated to an ON using a SMCC linker (e.g. PMO-CP8M); 
         FIG.  5   d    is a DCCPM of HP8M using a HNA linker (PMO-HP8M); 
       The FITC group here and elsewhere may be any other fluorescent label and is present merely to enable visualization; 
         FIG.  6    shows a general schematic of a FITC labeled DCCPM in which the n-termini of the FITC labelled PMO (compound I) and the CPA (compound III) are linked via a bi-functional linker disuccinimidyl glutarate (DSG) forming compound VI; 
         FIG.  7    shows a general schematic diagram of a DCCPM in which a FITC labelled PMO is directly conjugated to a CPA through a shared nitrogen forming compound VII; 
         FIGS.  8   a - 8   o    show the liquid chromatography-mass spectrometry conformation of the synthetic steps and the molecular masses of compounds depicted in  FIG.  1   ,  FIG.  2    and  FIG.  5   . All PMOs in this application are fluorescently labelled unless specified otherwise. 
         FIG.  8   a    is a LRMS (API-ES) mass spectra of CP8M-NC. Mass calculated for C 76 H 130 N 18 O 16 S 2+  (M+2H) 2+  791.5 found 791.8; 
         FIG.  8   b    is a LRMS (API-ES) mass spectra of CP8M. Mass calculated for C 74 H 125 N 18 O 16 S +  (M+H) +  1554.9 found 1554.5; 
         FIG.  8   c    is a LRMS (API-ES) mass spectra of CBM. Mass calculated for C 59 H 95 N 16 O 10 S +  (M+H) +  1220.5 found 1220.1; 
         FIG.  8   d    is a LRMS (API-ES) mass spectra of HP8M. Mass calculated for C 77 H 125 N 20 O 16   +  (M+H) +  1586.9 found 1586.5; 
         FIG.  8   e    is a HRMS (LQT-ESI) mass spectra of PMO. Mass calculated for C 332 H 500 N 153 O 110 P 25  (M) 9164.1675 found 9164.1882; 
         FIG.  8   f    is a HRMS (LQT-ESI) mass spectra of PMO-SMCC. Mass calculated for C 344 H 513 N 154 O 113 P 25  (M) 9383.2570 found 9383.2704; 
         FIG.  8   g    is a HRMS (LQT-ESI) mass spectra of PMO-CP8M. Mass calculated for C 418 H 638 N 172 O 129 P 25 S +  (M+H) +  10937.1806 found 10937.2377; 
         FIG.  8   h    is a HRMS (LQT-ESI) mass spectra of NF-PMO-CP8M. Mass calculated for C 393 H 619 N 172 O 121 P 25 S (M) 10490.0372 found 10490.2268; 
         FIG.  8   i    is a HRMS (LQT-ESI) mass spectra of PMO-HP8M. Mass calculated for C 417 H 626 N 173 O 127 P 25  (M) 10863.1285 found 10863.0716; 
         FIG.  8   j    is a LRMS (API-ES) mass spectra of FITC 3+ (CP8M-3). Mass calculated C 92 H 132 N 18 O 20 S 2+  (M+2H) 2+  920.5 found 921.0; 
         FIG.  8   k    is a LRMS (API-ES) mass spectra of FITC 2+ (CP8M-2). Mass calculated C 92 H 131 N 17 O 20 S 2+  (M+2H) 2+  913.0 found 913.6; 
         FIG.  8   l    is a LRMS (API-ES) mass spectra of FITC 1+ (CP8M-1). Mass calculated C 92 H 129 N 14 O 20 S +  (M+H) +  1783.1 found 1782.6; 
         FIG.  8   m    is a LRMS (API-ES) mass spectra of FITC+ (CP8M-0). Mass calculated C 92 H 126 N 11 O 20 S −  (M−H) −  1738.1 found 1738.0; 
         FIG.  8   n    is a HRMS (LQT-ESI) mass spectra of PMO-4-FB. Mass calculated for C 340 H 503 N 153 O 112 P 25   +  (M−H) +  9295.1802 found 9295.2168; and 
         FIG.  8   o    is a HRMS (LQT-ESI) mass spectra of PMO-CP8M-NC. Mass calculated for C 420 H 642 N 172 O 129 P 25 S +  (M−H) +  10965.2112 found 10965.2162; 
         FIGS.  9   a  and  9   b    demonstrate the comparative conjugation efficiencies of SMCC and HNA linker systems; 
         FIG.  9   a    shows the % Conversion of HP8M and PMO-4FB into PMO-HP8M analysed by UV spectroscopy. % Conversion was calculated by A 350  of the bis-aryl hydrozone bond based on the starting reaction concentration of 705 μM. UV profiles of corresponding starting materials (HP8M and PMO-4FB) and the resulting conjugate (PMO-HP8M); and 
         FIG.  9   b    shows the % isolated yield of PMO-CP8M and PMO-HP8M. 
         FIG.  10   a    shows florescence microscopy images demonstrating a dose dependent increase in DCCPM delivery into a human osteosarcoma cell line (U2OS) maintained in culture, without transfection reagent. The biologically active compound was a PMO with a sequence: 
       SEQ ID NO 1: 5′GGCCAAACCTCGGCTTACCTGAAAT3′ 
       (an antisense reagent targeted to exon 23 of the mouse dystrophin gene, that causes the exclusion of exon 23 during mRNA splicing maturation); the bi-functional linker was a PEGylated SMCC; and the StaP was RKF-S5-RLF-S5 (SEQ ID NO: 57). This configuration of DCCPM is depicted as PMO-CP8M in the subsequent figs; unconjugated PMO acts as a control. All compounds were added to U2OS cells maintained in culture, without transfection reagent for 4 hours; 
         FIG.  10   b    is a graphical representation of the delivery of PMO and PMO-CP8M into a human osteosarcoma cell line (U2OS) maintained in culture without transfection reagent; 
         FIG.  11    is the analysis of PMO uptake into HEK293T cells by flow cytometry. Cells were incubated without PMO or with 1 μM PMO, 1 μM PMO-C8M or 1 μM PMO-CP8M-NC (all fluorescein-labelled) at 37° C. for 4 hours. Fluorescence was measured after washing the cells with PBS. 
         FIG.  12   a    shows flow cytometry analysis of HEK293T cells treated with FITC+3 (CP8M3), FITC+2 (CP8M-2) and FITC+1 (CP8M-1) without transfection reagent for 4 hours; 
         FIG.  12   b    shows flow cytometry analysis of HEK293T cells treated with FITC+3 (CP8M3), FITC+2 (CP8M-2) and FITC+1 (CP8M-1) without transfection reagent for 4 hours. The graph represents mean fluorescent intensity of FL1; 
         FIG.  13    shows fluorescence microscopy images either 5 μM PMO-CP8M or 5 μM PMO delivery into a mouse cell line that harbours the mdx mutation of the dystrophin gene (H2K mdx) maintained in culture, without transfection reagent, in which the PMO has a fluorescent label. 
         FIG.  14    shows an agarose gel electrophoresis image demonstrating that exon exclusion of the mouse dystrophin exon 23 is restricted to H2K mdx cells that have been transfected with 5 μM PMO-CP8M but not those treated with 5 μM PMO alone, in which the PMOs have a fluorescent label. 24 hours after incubation with PMO-CP8M or PMO, H2K mdx cells were recovered and RNA isolated. The RNA was reversed transcribed and an amplification between exon 20 and 26 of the mouse dystrophin gene, followed by a nested amplification between exon 20 and 26 was performed to yield a full length product of 901 bp fragment if exon 23 is present or 688 bp fragment if exon 23 is excluded; 
         FIG.  15    shows immuno-cytochemical staining for dystrophin from Tibialis anterior muscles of mdx mice following a single intramuscular injection of either 2.2 nmol PMO-CP8M or 2.2 nmol PMO into the Tibialis anterior muscle (dose in respect of molarity of PMO). Muscles were recovered 7 day post administration; 
         FIG.  16    shows a graphical representation of the number of skeletal muscle fibre being positive for an immuno-cytochemical staining for dystrophin from Tibialis anterior muscles of mdx mice injected with either 2.2 nmol PMO-CP8M or 2.2 nmol PMO. All dose are given in respect of the molarity of PMO; 
         FIGS.  17     a - d  show immuno-cytochemical staining for dystrophin from skeletal muscles of mdx mice: 
         FIG.  17   a    is from the diaphragm following a single intraperitoneal injection of 1 μmol/kg PMO-CP8M or 1 μmol/kg PMO [* denotes non fluorescent labelled PMO]. Muscles were recovered 7 days post administration; 
         FIG.  17   b    is following multiple intravenous injections—total 4.4 μmol/kg PMO-CP8M or 4.4 μmol/kg PMO; 
         FIG.  17   c    is following multiple intraperitoneal injections—total 10.9 μmol/kg PMO-CP8M or 10.9 μmol/kg PMO; and 
         FIG.  17   d    is a graphical representation of the number of skeletal muscle fibre being positive for an immuno-cytochemical staining for dystrophin following intraperitoneal administration. All dose are given in respect of the molarity of PMO [* denotes non fluorescent labelled PMO]. Muscles were recovered 14 days post administration; 
         FIGS.  18   a - 18   c    show immuno-cytochemical staining for dystrophin from heart muscles; 
         FIG.  18   a    is of mdx mice following a single intraperitoneal injection of 1 μmol/kg PMO-CP8M or 1 μmol/kg PMO. Muscles were recovered 7 days post administration; 
         FIG.  18   b    is following multiple intraperitoneal injections—total 7.6 μmol/kg PMO-CP8M or 7.6 μmol/kg PMO. Muscles were recovered 14 days post administration; 
         FIG.  18   c    is following multiple intraperitoneal injections—total 10.9 μmol/kg PMO-CP8M or 10.9 μmol/kg PMO; 
         FIG.  18   d    is a graphical representation of the number of cardiac muscle fibre being positive for an immuno-cytochemical staining for dystrophin following a multiple intraperitoneal injections—total 7.6 μmol/kg PMO-CP8M or 7.6 μmol/kg PMO; and 
         FIG.  18   e    is a graphical representation of the number of cardiac muscle fibre being positive for an immuno-cytochemical staining for dystrophin following multiple intraperitoneal injections—total 10.9 μmol/kg PMO-CP8M or 10.9 μmol/kg PMO. All dose are given in respect of the molarity of PMO [* denotes non fluorescent labelled PMO]. Hearts were recovered 14 days post administration; 
         FIG.  19    shows immuno-cytochemical staining for dystrophin from heart muscles of mdx mice following intraperitoneal injections of PMO-CP8M with a total cargo size of 8.7 KDa and PMO-CP8M with total cargo size of 9.2 KDa. Hearts were recovered 14 days post administration; 
         FIG.  20    shows immuno-cytochemical staining for dystrophin from the cerebellum of mdx mice following a single intraperitoneal injection of 1 μmol/kg PMO-CP8M or 1 μmol/kg PMO. All dose are given in respect of the molarity of PMO [* denotes non fluorescent labelled PMO]. Cerebella were recovered 7 days post administration; and 
         FIG.  21    demonstrates in vivo liver cell uptake of fluorescein-labelled PMO after intravenous administration of PMO or PMO-HP8M to mdx mice (single intravenous injection, 1 μmol/kg, analysed 2 weeks post-injection). Un-injected mdx mice were used as a negative control. 
     
    
    
     DETAILED DESCRIPTION 
     The invention is illustrated with reference to a single example which proves the benefit of the claimed invention. 
     An exemplary drug carrying cell penetrating molecule (DCCPM) was produced with a FITC label in order to demonstrate cellular uptake (Example 1). 
     The exemplary DCCPM comprises:
         i) a biologically active compound (BAC)— (see Table 4 for non-limiting examples);   ii) a cell penetrating agent (CPA) which is a stabilized peptide (See Table 2 for non-limiting examples); and   iii) a bi-functional linker (BFL) (see Table 5 for non-limiting examples).       

     The three components forming the DCCPM are described in more detail below, although as illustrated in  FIG.  5   , the BAC and CPA can be linked directly ( FIG.  6   ). 
     1. The Biologically Active Compound. 
     The biologically active compound is any compound that can exert a biological effect within a biological cell. Preferably, though not essentially, the BAC is one which will impact on the expression of one or more endogenous or exogenous genes. Examples include nucleic acids, DNAzymes, ribozymes, aptamers and pharmaceuticals. Preferred biologically active compounds for use in the present invention include electrically neutral oligonucleotides (charge −1 to +1 at physiological pH—about 7.5) such as polynucleic acids (PNAs) or PMOs or their modified derivatives that might impart a small electric charge (either positive or negative). 
     The biologically active compound may be used as a steric blocking compound to suppress or enhance: i) RNA splicing; ii) protein translation or iii) other nucleic acid:nucleic acid or nucleic acid:protein interactions, altering the gene expression of endogenous or exogenous (pathogen derived) genes. 
     The hybridisation of ON&#39;s to specific RNA sequence motifs prevents correct assembly of the spliceosome, so that it is unable to recognise the target exon(s) in the pre-mRNA and hence excludes these exon in the mature gene transcript. Exclusion of an in-frame exon can lead to a truncated yet functional gene product; exclusion of an out of frame exon results in a frame-shift of the transcript, potentially leading to a premature stop codon and a reduction in the target gene expression level. 
     Additionally, ON&#39;s can be designed to target 5′ translation initiation start sites of endogenous or viral gene transcript(s) to prevent binding of the translational machinery. Using ASO to suppress viral translation is a well-established technology and has progressed into clinical trials for viral haemorrhagic fevers such as Marburg and Ebola. 
     Also, ON can be designed to target 3′ untranslated region of an endogenous transcript that alters the stability of the transcript. Such targets include, and are not limited to, poly adenylation and/or cleavage sites of the transcript. 
     Also, ON can be designed to form aptamers such that the secondary and tertiary structures can bind proteins or other cellular targets thus impacting on specific gene expression levels. 
     Non-limiting exemplary ON chemistries are illustrated in Table 4. 
     In the non-limiting example illustrated, the target is exon 51 of the dystrophin gene and comprises the sequence: 
     
       
         
           
               
               
            
               
                   
                 SEQ ID NO 2: 
               
               
                   
                 5′CUCCAACAUCAAGGAAGAUGGCAUUUCUAG3′ 
               
            
           
         
       
     
     2. The Cell Penetrating Agent (CPA) which is a Stabilized Peptide 
     The cell penetrating agents of the invention are stabilized peptides. 
     The peptides may be stabilized by stapling, to form a stapled peptide (StaP), or by stitching to form a stitched peptide (StiP) 
     All-hydrocarbon staples and stitches may confer a property, e.g. an α-helical structure, protease resistance, cellular penetrance, and biological activity. 
     Non-limiting examples of stapled and stitched peptide sequences are illustrated in Table 2 and include peptide sequences including S5, S8 and B5 (as defined in Table 2). 
     Stabilisation of e.g. the α-helical structure can be achieved by, for example, a ring-closing metathesis and may be catalysed by a variety of ruthenium catalysts including Grubbs generations 1 and 2 and Grubbs-Hoyveda generations 1 and 2. 
     All the peptide components (amino acids, unnatural amino acids, unstapled/unstitched, partially stapled/stitched and stapled/stitched peptides) may exist in specific geometric or stereoisomeric forms. All compounds include cis- and trans-isomers, (R)- and (S)-enantiomers, diastereoisomers and racemic mixtures thereof. 
     Preferred isomer/enantiomers will be enriched to give a greater proportion of one particular isomer or enantiomer. Embodiments thereof may be made of greater than 90%, 95%, 98% or 99%, by weight, of a preferred isomer/enantiomer. 
     Non-limiting examples of unnatural amino acids used in stabilising a peptide structure are illustrated in Table 1. 
     In one embodiment the applicant employs α,α-disubstituted unnatural amino acids bearing all-hydrocarbon tethers (e.g. α-methyl,α-pentenyl glycine). 
     For single turn stapling, one embodiment could employ a (S)-pentenylalanine (S5) at, e.g. i, i+4 positions, and in another embodiment, for double turn stapling, a combination of either R-octenylalanine/S-pentenylalanine (R8/S5) or S-octenylalanine/R-pentenylalanine (S8/R5) at e.g. i, i+7 positions can be used. The same pairings can be used to install more than one staple within a given peptide template. S5 can be substituted at i, B5 at position i+4 positions, and S8 can be been substituted at i, i+4, i+11 positions to generate stitched peptides. The S5 configured amino acid and its enantiomer R5, or S8 configured amino acid and its enantiomer R8, differ only in the opposite stereochemical configuration of the staple they bear. 
     Based upon the inclusion of a single or a double turn staple, peptides may comprise of one or more of the sequences in Table 2. Based upon the specific peptides shown in Table 2, a person skilled in the art can easily envisage peptides with 3, 4, 5 or more turn stabilising staples. 
     The hydrocarbon bridge may be composed of a double hydrocarbon bond or a single hydrocarbon bond. 
     In one embodiment the cell penetrating agent has a stitch or staple peptide comprising the sequence RFK-S5-RLF-S5 (SEQ ID NO: 57). 
     In another embodiment the peptide is a branched stapled peptide. The branched stapled peptide comprises of 2 or more chains of peptides. Branched peptides may be formed using any method know to the art; in one embodiment a lysine residue is used to branch two peptide chains. 
     Functional derivatives of disclosed peptide sequences could be used. Functional derivatives may have representative fragments or homologues or peptides that include insertions to the original peptide. Typical derivative would have 70%, 80%, 90% or more of the original peptide sequence and may have up to 200% of the number of amino acids of the original peptide. The derivatives would be used to enhance the delivery of a biologically active compound. 
     Peptide sequence can include modified amino acids to include functional groups that permit the addition of other moieties. Non-limiting examples of such moieties include an acetyl, a cholesterol, a fatty acid, a polyethylene glycol, a polysaccharide, an aminoglycan, a glycolipid, a polyphenol, a nuclear localising signal, a nuclear export signal, an antibody and a targeting molecule. 
     3. Bi-Functional Linker 
     A bi-functional linker may be used to link the BAC to the CPA. 
     Preferred linkers will link between, for example, an amine group on the BAC and a sulfhydryl (thiol) group (usually a cysteine residue) on the CPA terminus. Examples of substrates to achieve this include, but are not limited to, SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate), AMAS (N-α-maleimidoacet-oxysuccinimide ester, BMPS (N-β-maleimidopropyl-oxysuccinimide ester), GMBS (N-γ-aleimidobutyryl-oxysuccinimide ester), DMVS (N-δ-maleimidovaleryl-oxysuccinimide ester, EMCS (N-ε-malemidocaproyl-oxysuccinimide ester), and LC-SMCC (Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate) as exemplified in Table 5. 
     Another preferred linker system is hydrazynal nicotinic acid (HNA), however if the BAC is a PMO, the PMO is modified to incorporate 4 formyl benzioic acid. 
     Other linkers such as DSG (disuccinimidyl gluterate) and DSCDS (disuccinimidyl-cyclohexl-1,4-diester) will include the ability to link the 5′-amino group of the BAC to the N-terminus of the CPA (Table 5, entries 8 and 9). 
     Linkers may include other elements that confer a desirable property on the DCCPM e.g. spacer between ON and CPA or an element that will enhance solubility, for example a PEGylated element as illustrated in  FIGS.  5   c  and  5   d   . Non-limiting examples are shown in Table 5. 
     The biologically active compound is covalently attached to the chimeric cell delivery peptide. Again, this can be done using any method known in the art. Preferably, the cell delivery peptide is attached to the biologically active compound by means of a disulphide bridge or a thiol maleimide linker e.g. SMCC; the attachment may be by means of an amide linker or an oxime linker or a thioether linker. 
     Example 1: (Proof of Principle) 
     DCCPM to Enhance RNA Steric Blocking in Treating Duchenne Muscular Dystrophy (DMD). 
     INTRODUCTION 
     Duchenne muscular dystrophy (DMD) is the most common inherited lethal childhood disease in the world, with a worldwide incidence of approximately 1 in 4000 live births 33 . This severe muscle-wasting disorder is caused in the majority of families by gene mutations leading to disruption of the reading frame and premature truncation of the protein dystrophin 34,35 . 
     RNA splicing suppression of the DMD transcript has particular promise. The hybridisation of ASOs to specific RNA sequence motifs prevents correct assembly of the spliceosome, so that it is unable to recognise the target exon(s) in the pre-mRNA and hence excludes them in the mature gene transcript. ASO-mediated RNA splicing suppression resulting in the re-expression of a truncated, yet functional dystrophin protein has been demonstrated in vitro and in the pre-clinical mdx mouse model 29,36-41 , which led to clinical development programs 8,13 . 
     Although intravenously administered PMOs have demonstrated a dose-dependent increase in dystrophin re-expression with some functional benefit 13,42 , skeletal muscle dystrophin restoration is still very variable between patients after many multiple administrations. Importantly, many other target tissues (e.g. brain and heart) remain refractory to PMO transfection even when repeat administration or high dose strategies are employed 27-29 . 
     To date unmodified CPA conjugation improves PMO bio-distribution and serum stability 30-32 , however toxicity is still a major roadblock for pipeline development 19 . 
     Applicant hypothesised that a CPA based upon a stabilized e.g. StaP (or StiP) conjugated to a PMO known to cause RNA splicing suppression of the DMD transcript, would lead to a greater level of dystrophin restoration and re-expression of dystrophin in tissues refractory to naked PMO without the potential for CPA related toxicity. 
     Materials and Methods 
     Nuclear Magnetic Resonance (NMR) Analysis of StaP 
       1 H NMR spectra was recorded using a Bruker Avance III 500 (500 MHz) spectrometer. Samples were dissolved in H 2 O with 10% D 2 O and 10 mM sodium acetate. 
     NOESY spectra were recorded with a 12626.263 Hz sweep width, 4096 complex points (DQD acquisition mode) in the direct dimension and 1024 indirect points (States-TPPI acquisition mode). A NOESY mixing time of 250 ms was used to provide cross peaks with high signal to noise while largely avoiding spin diffusion. A pre-saturation pulse on water and a 3-9-19 pulse sequence with 20% Z-gradients (4,5) aided solvent suppression. TOCSY spectra were recorded with the same spectral width and resolution as the NOESY with a homonuclear Hartman-Hahn transfer using the MLEV17 sequence for an 80 ms mixing time (6). Two power levels were used for excitation (3 dB) and spinlock (12.2 dB). Water suppression was achieved as with the NOESY. 
     High Resolution Mass Spectroscopy 
     High-resolution mass spectra were recorded on a Thermo scientific LQT Orbitrap XL under electron spray ionization conditions (ESI) or where indicated under Atmospheric Pressure Ionisation (API) condition. 
     Circular Dichroism (CD) Spectroscopy 
     CD analysis was performed on an Applied Photophysics Chirascan Circular Dichroism spectrometer. Samples were dissolved in D 2 O at 0.125 W/W % and data acquired in triplicate at room temperature and subsequently averaged and smoothed using built in qCD software. Graphs were plotted by subtracting a blank D 2 O spectrum from the acquired data to provided blank correction. 
     Synthesis of PMO-CP8M and NF-PMO-CP8M 
     PMO (22.2 mg, 2.1 μM) was dissolved in PBS (400 μL, 1 x) and incubated at room temp after the addition of SMCC linker (6 mg, 18 μM, 9x excess) dissolved in MeCN 100 μL. After 45 mins the mixture was desalted using sephadex g25 hydrated in a PBS 1x and was also used as the eluent. RCM-C-PEG-8Mer (3 mg, 2.5 μM) was mixed immobilised TCEP (750 μL) for 1 h. The SMCC modified PMO was then desalted into PBS/MeCN (500 μL 4:1) and immediately the peptide was eluted from the immobilised TCEP and stirred at room temp for 3 hours before purification on a Waters HLB column. 
     The solution was loaded onto 4 HLB columns, and washed with milliQ water to remove any salts then 20% MeCN in water and finally PMO-CP8M was removed with 50% MeCN in water. The MeCN content was reduced by rotary evaporation and the conjugate subsequently freeze dried to yield the final lipholysed compound. 
     Synthesis of PMO-HP8M 
     Modification of PMO to PMO-4FB. 
     4-FB (250 mg, 1.5 mM) was dissolved in DMF with COMU (1.2 g, 2.6 mM) and NHS (230 mg, 2.0 mM) and stirred for a few mins. Nb, 4-FB did not fully dissolve until DIEA was added. DIEA (0.54 mL 3.0 mM) was then added upon which the reaction mixture changed from colourless to pale yellow/orange. The reaction mixture was stirred for 1 h and monitored by TLC using 5% MeOH in DCM. The mixture was separated over DCM to remove DMF then purified by flash chromatography using DMC to elute the top spot staining positive with 2,4 DNP. Product was collected as an off white solid 112 mg (30%). 
     PMO (30.4 mg, 3 μM) was added to a solution of 4-FB and dissolved in Carbonate buffer:MeCN (50% MeCN) and NHS activated 4-FB (10 mg, 32 μM) was added and stirred overnight. The mixture was then desalted using sephadex G25 superfine with water:MeCN as an eluent. MeCN was removed by rotary evaporation and the remaining eluent was then freeze dried. Freeze dried product yielded 24 mg 83% yield. 
     Conjugation of PMO-4FB to HP8M 
     HP8M was dissolved in milliq ultra pure water (100 μL) to give a solution of 12 mg/mL. Aldehyde modified PMO (7 mg, 0.76 μM) was dissolved in water/MeCN (300 μL, 1:1) and desalted using sephadex G25 superfine and water/MeCN (1:1) as the eluent. The collected fraction was then diluted to 1 mL total volume in water:MeCN mix (1:1) and PMO content was analysed by UV/vis and found to be 6.5 mg/mL or 705 μM. HNA peptide and Analine (10 mM final conc) was then added and UV/vis monitored for evidence of A 354  and used to calculate the conjugation of PMO to peptide. 
     PMO and Peptide Synthesis 
     PMO were synthesised with a 5′ amine group and 3′ fluorescein isothiocyanate (FITC) label and purified &gt;90% by Genetool LLC (Philomath, Oreg. USA). All peptides were synthesized following an established protocol using standard Fmoc-peptide chemistry on Rink amide MBHA resin. The coupling reactions were performed by the addition of a mixture of 10 equivalents of the amino acids, 9.9 equivalents of HCTU and 20 equivalents of DIPEA in NMP (equivalents relative to initial loading of Rink amide MBHA resin). The reactions were allowed to proceed for at least one hour. Coupling of non-natural amino acids (R/S5, R/S8 or 85) was performed with 4 equivalents of the amino acid, 3.9 equivalents of HCTU and 10 equivalents of DIPEA in NMP for two hours. The ring closing metathesis reaction of the olefin-containing non-natural amino acids was facilitated with Grubbs|catalyst (benzylidene-bis(tricyclohexylphosphine)-dichlororuthenium) dissolved to approximately 10 mg/mL in 1,2-dichloroethane (DCE) for two hours under nitrogen bubbling. Subsequently, excess catalyst was washed from the resin with DCE and then coupled with an N-terminal FITC. Upon completion, peptides were simultaneously cleaved from the resin and de-protected using a cleavage cocktail containing 95% TFA, 2.5% TIS and 2.5% water. Crude peptides were dissolved in 50% acetonitrile/water, passed through a 0.2 μm syringe filter, and purified by reverse phase HPLC using a C-18 column (Agilent, Palo Alto, Calif.). Compound identification and purity was assessed using coupled LC/MS (Agilent, Palo Alto, Calif.). Purified fractions were pooled and evaporated to remove acetonitrile and trace TFA by Speedvac and then lyophilized to dryness. A non-ring closed peptide was also produced as a control. 
     Cell Culture and Transfection 
     U2OS cells (Human osteosarcoma) were cultured in high glucose DMEM supplemented with 10% foetal calf serum (Sigma, UK) at 37° C. under an 8% CO2/92% air atmosphere. 
     H 2 K mdx mouse myoblasts were cultured at 33° C. under a 8% CO 2 /92% air atmosphere in high-glucose DMEM supplemented with 20% foetal calf serum, 0.5% chicken embryo extract (PAA laboratories Ltd, Yeovil, UK), and 20 units/ml γ-interferon (Roche applied science, Penzberg, Germany). Cells were then treated with trypsin and plated at 8×10 4 /cm 2  in 24-well plates coated with 0.1 mg/ml ECM gel (Sigma). H 2 K mdx cells were transfected 24 h after seeding with treatment in a final volume of 0.2 ml of normal growth media. Following 4 hours of transfection, the PMO or PMO-SAP was removed and replaced with DMEM supplemented with 5% horse serum. Fluorescence and RNA extraction was performed 48 hours post transfection. 
     HEK293T cells (Human embryonic kidney) were cultured in high glucose DMEM supplemented with 10% foetal calf serum (Sigma, UK) at 37° C. under an 8% CO 2 /92% air atmosphere. 
     U2OS cells were incubated with PMO or PMO-CP8M at increasing concentration (0.5 μM, 1.0 μM, 5.0 μM and 10 μM) with any facilitation transfection reagent; H2K mdx mouse myoblasts were incubated with PMO or PMO-CP8M at 5.0 μM: HEK293T cells were incubated with CP8M (1.0 μM, 10 μM and 100 μM) and PMO, PMO-CP8M-NC, PMO-C8M at 1.0 μM. Levels of fluorescence was quantified at 494 nm to determine relative entry of respective compounds by microscopic or flow cytometry methodologies. 
     RNA Extraction and Nested RT-PCR Analysis 
     Total RNA was isolated from H 2 K mdx mouse myoblasts cells (RNeasy, Qiagen, UK). The RNA was reversed transcribed (nanoscript2, Primer Design UK) and an amplification between exons 20 and 26, followed by a nested amplification between exon 20 and 26 was performed to yield a full length product of 901 bp or 688 bp if the mouse dystrophin exon 23 was excluded. Products loaded in a 1% agarose gel (buffered with tris acetate 40 mM and 1 mM ethylenediaminetetraacetic acid). 
     Animals 
     mdx mice, with access to chow and water ad libitum, were used in all experiments. All experiments were carried out in the Animal unit, School of Biological Science, University of Reading, Reading, UK according to procedures authorized by the UK Home Office. Mice were killed by CO 2  inhalation or cervical dislocation at desired time points, and muscles and other tissues were snap-frozen in liquid nitrogen-cooled isopentane and stored at −80° C. 
     Administration of PMO or PMO-CP8M 
     Intramuscular administrations: Tibialis anterior muscles of mdx mice were injected with either 2.2 nmol PMO or 2.2 nmol PMO-CP8M under isoflurane anaesthesia. Systemic administration: mdx mice were subject to a single or repeated intraperitoneal injections of PMO or PMO-CP8M (or non-fluorescently labelled variants) at doses ranging from 1 μmol/kg to 10.9 μmol/kg total delivery; alternatively, mdx mice were subject to single or repeated intravascular injections of PMO or PMO-CP8M ranging from 1 μmol/kg to 4.4 μmol/kg total. A series of tissues were recovered at the end of the experiment that included skeletal muscle, heart, brain and liver. 
     Histology and Immuno-Cytochemistry 
     For skeletal muscle, heart and brain, 10 μM cryosections were cut and dystrophin protein was detected using rabbit polyclonal antibody to dystrophin (ab15277; Abcam, Cambridge, UK). Routine haematoxylin and eosin staining was used to assess general pathology and morphology. For liver, 10 μM cryosections were dried and embedded in fluorescence-compatible mounting medium (Dako), and general fluorescence was assessed microscopically at 494 nm. 
     Flow Cytometry 
     Uptake of fluorescently-labelled PMO was determined by flow cytometry using an Accuri C6 flow cytometer. PMO-transfected cells were released with trypsin, washed in PBS and kept on ice before analysis. Cell fluorescence in single live cells was determined using FlowJo software after appropriate gating. Untreated cells were used to establish gating settings for the determination of the % fluorescein-positive cells. 
     Statistical Analysis 
     All data are reported as mean values±SEM. Statistical differences between treatment groups and control groups were evaluated by SigmaStat (Systat Software, UK) and student&#39;s t test was applied. Significance was accepted for p-values&lt;0.05. 
     Results 
     Circular dichroism and the nuclear magnetic resonance data confirmed that the ordered structure of the peptides was as expected and that the stapled peptides adopted an α-helical structure ( FIG.  4   ). 
     The conjugation of PMO-SMCC with a CPP to form PMO-CP8M has consistently yielded an efficiency of 10%. Surprisingly, adopting a conjugation based upon a PMO modified to incorporate 4 formyl benzioic acid and hydrazynal nicotinic acid (HNA) incorporated into the terminal end of the CPP, increased the efficiency of conjugation to yield PMO-HP8M at 59% ( FIG.  9   ). 
     PMO was conjugated to the bi-functional linker (a PEGylated SMCC) and a CPP (RKF-S5-RLF-S5 (SEQ ID NO: 57)) as confirmed by mass spectrometry ( FIG.  7   ). Subsequently PMO and PMO-CP8M were transfected into a standard cell line (U2OS) to determine if the CP8M conferred enhanced cell entry to the cell. Naked PMO were refractory to cell entry, giving only a background fluorescence signal, compared to a dose dependent increase in fluorescence with PMO-CP8M ( FIGS.  10   a  and  10   b   ). The lack of signal above background does not allow statistical analyses of the comparative increase in fluorescence, but clearly demonstrated that without the CP8M conjugation, PMO did not enter the cell. 
     Transfection experiments conducted in the HEK293T human embryonic kidney cells again demonstrated that PMO was refractory to cell entry. Importantly, a non-ring closed variant of CP8M (termed CP8M-NC) also did not result in significant cell entry above that of PMO alone. However a ring closed variant that contains the core sequence RKF-S5-RLF-S5 (SEQ ID NO: 57) demonstrated that when conjugated to a fluorescently labelled PMO (to form PMO-C8M), that the PMO was now efficiently taken into cells ( FIG.  11   ). This confirms that the shape imposed upon the peptide sequence, following a ring closing metathesis, is important to facilitate cellular entry of a DCCPM in which the BAC is an ON, more specifically a PMO. 
     The formal charge of CP8M is +3 at physiological pH. We also provide data demonstrating that reducing the formal charge within this sequence still leads to a surprising and significant cellular entry of CP8M variants with formal charges of +2 (CP8M-2) and +1 (CP8M-1) ( FIGS.  12   a  and  12   b   ), particularly at lower concentrations. The reduction in charge leads to solubility issues which account for the failure of a dose dependent increase, unlike that observed with CP8M. Alternative excipients or manipulations of the peptide as highlighted in Table 5 and Table 6 are likely to overcome these solubility issues. 
     When transfection experiments were conducted in the H2K mdx mouse myoblasts cells, it confirmed the finding that PMO are refractory to muscle cell entry, which was overcome with the conjugation of CP8M ( FIG.  13   ) and that the CP8M mediated delivery of PMO resulted in the steric blockade of RNA editing of the dystrophin transcript, such that exon 23 was excluded from the transcript ( FIG.  14   ). Again, the lack of exon exclusion from the PMO only samples precludes comparative statistical analyses; but highlights that exon exclusion, resultant from entry of a PMO, only occurs when the PMO is conjugated to CP8M. 
     In order to determine if CP8M hindered the biological activity of the PMO, direct intramuscular administrations (2.2 nmol) were conducted into the Tibialis anterior muscle of mdx female mice, with muscle recovered 7 days post-administration. The percentage of dystrophin re-expression was equivocal between the PMO-CP8M (805.75) and the naked PMO (762.25) with no statistical significant difference (n=4, p=0.863;  FIG.  15    and  FIG.  16   ). Thus it was determined that CP8M does not confer any steric hindrance to the biological activity of the PMO. 
     Systemic administrations of PMO-CP8M and PMO were conducted in mdx mice to determine if the CP8M moiety enhanced cell entry into skeletal muscle. Varying sub-optimal amounts (totally 1 μmol/kg, 4.4 μmol/kg, 10.9 μmol/kg) were administered by either intraperitoneal or intravenous injections, and diaphragm and/or Tibialis anterior (TA) muscle recovered 7 days post-administration. After a single intraperitoneal administration of 1 μmol/kg the diaphragm muscle gave more dystrophin positive fibres after PMO-CP8M treatment compared to the PMO control group ( FIG.  17     a, n= 1). In tibialis anterior muscles, intravenous (4.4 μmol/kg; n=3) data was equivocal between groups ( FIG.  17   b    and  FIG.  17   d   , p=0.201), as was the intraperitoneal (10.9 μmol/kg; n=4) data ( FIG.  17   c    and  FIG.  17   d   , p=0.886). 
     (4.4 μmol/kg; n=3) data was equivocal between group ( FIG.  17   b    and  FIG.  17   d   , p=0.201), as was the intraperitoneal (10.9 μmol/kg; n=4) data ( FIG.  17   c    and  FIG.  17   d   , p=0.886). 
     Systemic intraperitoneal administrations of PMO-CP8M and PMO were conducted in mdx mice to determine if CP8M enhanced cell entry ( FIGS.  18 - 21   ). A series of tissues was recovered and frozen 1 or 2 weeks post-administration (skeletal muscle, heart, brain and liver). 
     A single low dose of PMO or PMO-CP8M (1 μmol/kg) was administered (n=1 per group) and tissues recovered 7 days post-administration. Dystrophin-positive heart muscle fibres were detected after PMO-CPM8, but not PMO administration ( FIG.  18   a   ). 
     In addition, we carried out repeated intraperitoneal administrations into mdx mice. Intraperitoneal injections of PMO-CP8M (without fluorescent label) totaling 7.6 μmol/kg over 4 days (n=4 per group) lead to a significant increase in dystrophin-positive heart muscle fibres 2 weeks post-administration compared to injection of an equimolar amount of PMO (340±69 vs 57±17 fibres, p&lt;0.01;  FIGS.  18   b    &amp;  18   d ). 
     Furthermore, Applicant carried out intraperitoneal administration of PMO or PMO-CP8M, totaling 10.9 μmol/kg over 2 days (n=4 per group). Again, administration of PMO-CP8M lead to a significantly higher number of dystrophin-positive heart muscle fibres compared to administration of PMO (680±163 vs 33±8 fibres, p&lt;0.05;  FIGS.  18   c    &amp;  18   e ). 
     PMO-peptide conjugates of both 8.7 kDa and 9.2 kDa were successfully delivered to heart muscle fibres as evidenced by the induction of dystrophin re-expression in the hearts of mdx mice ( FIG.  19   , n=4). 
     A single low dose (1 μmol/kg) intravenous administration of PMO-CP8M, but not PMO (both without fluorescent label), led to recovery of dystrophin expression in the Purkinje cells of mdx mouse cerebellum ( FIG.  20   ; n=1 per group). No dystrophin expression was observed in the PMO control. 
     A single low dose (1 μmol/kg) intravenous administration of PMO-HP8M led to increased hepatocyte fluorescence in the vicinity of blood vessels two weeks post-administration compared to administration of PMO ( FIG.  21   ; n=1 per group), implying increased uptake of PMO-HP8M in hepatocytes. 
     CONCLUSION 
     From the data generated it can be seen that the conjugation of a CPA, stabilized by stapling, to a BAC (in the form of a PMO), via a BFL, facilitates entry of the PMO into a cell. The StaP CPA facilitated PMO entry in both in vitro and in vivo assay systems. 
     Applicant&#39;s data presents evidence that modified linker systems based on HNA and 4 formyl benzioic acid improve the efficiency of conjugation between a BAC and CPA. 
     Surprisingly, variants of CPA in which the formal charge is reduced demonstrate enhanced cell entry at lower concentrations. This will have important sequelae with respect to improving the toxicological profile of CPA, more specifically a CPP. 
     The in vivo model of RNA splicing suppression demonstrated that the biological action of an α-helical peptide conjugated PMO is equivalent to naked PMO following intramuscular administration, thus determining that no steric hindrance is exerted upon the PMO when coupled to an α-helical peptide moiety. 
     The data demonstrates the fact that in the in vivo model of RNA splicing suppression the stabilized CPA may enhance cell entry into skeletal muscle, particularly at lower doses. However PMO are known to enter skeletal muscle without a CPP conjugation. 
     Surprisingly, and very significantly, it has been demonstrated that tissues refractory to naked PMO transfection re-express dystrophin protein in both the heart and brain (purkinje cell) compartments when the PMO is conjugated with a StaP. 
     Applicant further provides evidence that the CPP can facilitate the entry of cargoes of different size and mass far beyond that stated in the current state of the art. 
     The repertoire of human and animal diseases that can be addressed is now expanded and enhanced due to the increased pharmacodynamics of the PMOs when conjugated with a stabilised peptide. Neuromuscular disease, metabolic disease, cancer, age-related degenerative diseases and acquired viral infection can all be targeted. 
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