WO2011020188A1 - Peptide sequences and peptide-mediated sirna delivery - Google Patents

Abstract

Peptides and a complex comprising a peptide and a cargo molecule, wherein the peptide and the cargo molecule are coupled by a non-covalent molecular association Peptides of the invention are designed according to five separate design criteria Certain members of these groups w ere found to facilitate the delivery en of siRNA molecules into eukariotic cells and siRNA mediated silencing of cellular targets

Description

PEPTIDE SEQUENCES AND PEPTIDE-MEDIATED siRNA DELIVERY

This application claims priority to United States provisional patent application 61/235,934 filed August 21, 2009.

FIELD OF THE INVENTION The present invention relates to peptide based siRNA delivery.

BACKGROUND OF THE INVENTION

RNA interference (RNAi) is a form of post transcription gene silencing, where the target mRNA is degraded prior to translation, thus the expression of the encoded protein is prevented. siRNAs are small RNA duplexes of 21-23 nucleotides capable of inducing the RNAi process in the cytosol. The major limitations for the use of siRNA both in vitro and in vivo are the instability of naked siRNA in physiological conditions and the bloodstream, and the inability to cross the cellular membrane to gain access to the intracellular environment.

When administered, a significant portion of nucleic acids (NAs) are excreted through the reticuloendothelial system (RES) mainly due to their small size and hydrophilicity. Moghimi and Bonnemain described a possible transfer passage through fenestrae in the endothelium of the liver for small particles (less than 100 nm) [Moghimi, et al. 1999]. It was also reported that highly charged particles can be recognized by the RES more rapidly than neutral or slightly charged particles [Benoit, et al. 2006, Mahato. 2005]. Furthermore, NAs are subjected to enzymatic degradation during circulation and within the cell. As a result, the potency of the administered NA is decreased, and in some cases an increase in dosage is required to compensate these effects.

Recently, the carrier-mediated delivery system has become a prevalent approach for improving the cellular uptake of siRNA. The carriers are designed to enhance cell targeting, prolong circulation time, and improve membrane permeation, while being biocompatible and biodegradable.

Many delivery systems, including lipids, peptides, synthetic and natural polymers, gold or virus capsids in the form of liposomes, micelles, emulsions, microemulsions, micro/nano-tubes, have been proposed with the goals to improve stability, transport, targeting and efficacy of siRNA therapeutics. However, most of these systems suffer from high toxicity and low transfection efficiency, especially for non-viral delivery. Techniques such as electroporation and microinjection have been developed to deliver different cellular effectors into cells, but most of these techniques are invasive.

Certain peptides have been demonstrated to translocate across the plasma membrane of eukaryotic cells.

Specific cell-penetrating peptides (CPPs) identified as useful as carriers for NAs have been described, see e.g. International patent applications publication nos. WO2007/076904 to Brock et al. and WO2007/069090 to Divita et al., although not all describe the transport of siRNA, see e.g. United States patents 7,163,695 to Mixson and 7,112,442 to Rice et al.. In many of these carrier-mediated delivery systems the NA is covalently linked to a carrier peptide of a specific sequence, see e.g. International patent application publication no. WO2008/063113 to Langel et al. and United States patent application publication no. US2005/0260756 to Troy et al. Specific peptides have been linked to NA via chemical linkers, see e.g. WO2008/033285 to Troy et al and WO2007/069068 to Alluis et al. United States patent 7,420,031 to Karas reports a peptide capable of delivering NAs to an intracellular compartment of a cell; the peptide-cargo moiety complex is formed by a chemical cross-linking or bridging method. United States patent 7,306,784 to Piwinica- Worms describes use of cell membrane-permeant peptide conjugate coordination and covalent complexes having target cell specificity. International patent application publication no. WO2003/106491 to Langel et al. describes methods for predicting, designing, detecting, and verifying CPPs and their use for improved cellular uptake of a cellular effector coupled to the CPP.

United States patent application publication no. US2008/0234183 to Hallbrink et al. describes methods for predicting, designing, detecting and verifying CPPs. The CPP-cargo complexes of Hallbrink et al. involve a covalent linkage between the CPP and cargo molecule.

United States patent 6,800,481 to Holmes et al. describes the self-assembly of amphiphilic peptides, i.e., peptides with alternating hydrophobic and hydrophilic residues, into macroscopic membranes. International patent application publication no. WO2007/089607 to Rana relates to the synthesis and formulation of nanotransporters for use as delivery agents for NAs and/or pharmaceutical agents. The nanotransporters have a central core which is a nanoparticle (e.g. a polylysine dendrimer) or a nanotube. Despite the versatility and biodegradability of the peptide delivery system, its use for

NA delivery has so far been limited by its low transfection efficiency when compared to lipid and virus based delivery systems.

SUMMARY OF THE INVENTION

In one embodiment, there is provided a complex comprising a peptide and a cargo molecule, wherein the peptide and the cargo molecule are coupled by a non-covalent molecular association.

In another embodiment, there is provided a complex comprising a peptide and a cargo molecule, wherein the peptide has an amino acid sequence selected from the group comprising: n-RLLRLLLRLWRRLLRLLR-c (SEQ. ID. NO 1 ) n-FQFNFQFNGGGHRRRRRRR-c (SEQ. ID. NO 2) n-ACSSSPSKHCGGGGRRRRRRRRR-c (SEQ. ID. NO 3) n-HRLRHALAHLLHKLKHLLHALAHRLRH-c (SEQ. ID. NO 4) n-LRHLLRHLLRHLRHLLRHLRHLLRHLLRH-c (SEQ. ID. NO 5) n-RFTFHFRFEFTFHFE-c (SEQ. ID. NO 6) n-LAELLAELLAELGGGGrrrrrrrrr-c (SEQ. ID. NO 7).

In another embodiment, there is provided a peptide having an amino acid sequence selected from SEQ. ID. NO 1 to SEQ. ID. NO 7.

In another embodiment, there is provided a complex comprising a peptide and a cargo molecule, wherein the peptide has an amino acid sequence of any one of SEQ ID NOs: 8 to 957. In another embodiment, there is provided a peptide having an amino acid sequence selected from SEQ. ID. NO 8 to SEQ. ID. NO 957.

BRIEF DESCRIPTION OF THE FIGURES

These and other features of the preferred embodiments of the invention will become more apparent in the following detailed description in which reference is made to the appended drawings wherein:

Figure 1. FACS results for the untreated cells, cells treated by peptide/siRNA, or

Lipofectamine/siRNA in 48 hours. Cell line: C166-eGFP. Figure IA is Peptide Cl. Figure

IB is Peptide C6. Figure 1C is Peptide A7. Figure ID is Peptide Al l. Figure IE is Peptide Bl . Figure 1 F is Peptide E3.

Figure 2 illlustrates the silencing efficacy of positive control and Peptide-siRNA complexes in C166-GFP cell line.

Figure 3 illustrates the transfection efficacy of promising peptides in comparison with

Lipofectamine 2000™ in Shh-Light II cells.

Figure 4 illustrates the cytotoxicity of peptides only and peptides/siRNA complexes against

C166-GFP cells.

Figure 5 illustrates the DMSO concentration effect on the toxicity of the solution against

C166-GFP cells.

Figure 6 illustrates the effect of serum and siRNA concentration on the transfection efficiency in the C166-eGFP cells. Figure 6 A shows the serum effect. Figure 6B shows the siRNA concentration effect.

DETAILED DESCRIPTION OF THE INVENTION

The rationale for peptide mediated NA delivery evolved from the biochemical knowledge that the active sites of enzymes, receptor ligands and antibodies involve about 5 to 20 amino acids, which may provide peptides with active targeting capability. This class of peptides may include protein-derived cell penetrating peptides (CPPs) or protein transduction domains (PTDs) [Langel. 2007, Deshayes, et al. 2005], cationic peptides [Fuchs, et al. 2006], and designed amphiphilic cell penetrating peptides [Niidome, et al. 1999].

The present invention provides novel peptides possessing both siRNA-co-assembling and cell penetrating properties. In one embodiment, the present invention provides new co-assembly peptides sequences that can promote siRNA transfection efficiency. The sequences were identified by means of a designed library of peptide sequences based on several parameters, which was screened to evaluate peptides efficacy to deliver siRNA and knock down the gene of interest in different cell lines. Transfection experiments with peptides of the present invention resulted in equal or better silencing percentage compared to that of Lipofectamine 2000™. Lipofectamine 2000™ available from Invitrogen is a cationic-lipid transfection reagent considered an industry standard in this field. These sequences also showed serum stability and less toxicity, which are challenging problems in lipid-based drug/gene delivery systems. The peptides of the present invention are more biocompatible and less toxic, compared to Lipofectamine 2000™.

The peptides of the present invention exhibit co-assembly capability, i.e. peptide- siRNA complexation. This assembly property plays an important role in enhancing the peptide/drug formulation and its efficiency. The peptides may also exhibit self-assembly which may be advantageous. For example, it may enhance the complex stability.

In carrier mediated NA delivery, the carrier-NA nanoparticles can be formed by conjugation where the two molecules are covalently linked together. Several options are available for covalent conjugation of peptides to NAs, including the use of cross-linking agents, triplehelix-forming oligonucleotides, and chemical attachment to the ends of linear NAs [Langel. 2007, Meade, et al. 2007]. Often conjugation of peptides to NAs involves formation of a disulfide bond [Hallbrink, et al. 2001], which can be rapidly cleaved in the reducing environment of the cell. In contrast, covalent bonds between the carrier and siRNA, such as conjugation, and the use of cross-linking agents, increase the complexity of the design and processing of the siRNA formulation. Such complexes may also have difficulty at releasing the siRNA at the target because of the strong bonding. Molecular association usually driven by weak interactions, such as hydrogen bonding, electrostatic or hydrophobic interactions have been used to form carrier-drug nanoparticles [Deshayes, et al. 2008, Crombez, et al. 2007, Veldhoen, et al. 2006].

In comparison with chemical modifications of the phosphodiester backbone, which is time-consuming and costly, carrier-drug complexed by weak bonding of peptide to NA provides a simple and fast means to formulate NA therapeutics and protect the NAs from degradation. Many positively charged peptides can interact with the negatively charged phosphate backbones of NAs through electrostatic interactions. The stoichiometry of complexation/assembly may affect the formulation and subsequent cellular uptake In one embodiment, the present invention provides complexes of co-assembly peptides of the present invention and cargo molecules. In a preferred embodiment, the cargo molecules of the present invention are NAs and, in a particularly preferred embodiment, the cargo is siRNA. Advantageously, the peptide-cargo complexes of the present invention are coupled by a non-covalent molecular association, which provides a simple and fast means to formulate siRNA therapeutics and protect the siRNAs from degradation as compared to prior art complexes involving covalent bonds.

The molecular association of the complexes of the present invention is also less expensive and complex than cross-linking or bridging methods. Through this assembly, peptide-siRNA complexes/assemblies, often in the form of nanoparticles, can be conveniently generated.

In one embodiment, the present invention provides peptides having one of amino acid sequences SEQ ID NOs. 1 to 7 shown in Table 1.

The leucine in SEQ ID NO 1 can suitably be replaced with other hydrophobic residues, such as isoleucine, phenylalanine or alanine without altering the helical structure of the original sequence based on secondary structure prediction and accordingly, has similar siRNA-co-assembly properties. Accordingly, in one embodiment, the present invention provides peptides having one of amino acid sequences SEQ ID NOs. 8 to 10 shown in Table 2.

The phenylalanine in SEQ ID NO 2 can suitably be replaced with other hydrophobic residues such as alanine, valine, leucine or isoleucine. Further, the glutamine and arparagine hydrogen bonding pair can be replaced with other hydrogen bonding pairs, such as N-S, N-T, H-T, Q-H, Y-Q or Y-H and the position of the members of the hydrogen bonding pairs can be switched. In the self-assembling region, the change of hydrogen bonding pairs and hydrophobic pairs will not significantly alter the siRNA loading region (multiple R region). In addition, these sequences remain amphiphilic in the self-assembling region. Accordingly, these sequences will have similar siRNA loading and transfection abilities as SEQ ID NO 2. Further arginine can be replaced with its D-form. The D-form of R (r) has been reported to enhance cell penetration ability, something also demonstrated by the present inventors with respect to SEQ ID NO 7. Further, the number of glycine, which functions as a spacer can be varied between 0 to 4 without significantly affecting the siRNA co-assembly property and transfection capability since this region is not involved in siRNA co-assembly and peptide self-assembly. The number of glycine is preferably between 0 and 4 because a shorter linker reduces the difficulty and cost of peptide synthesis. However, 5 or even more glycine may be used for a functional peptide and selecting the appropriate number would be within the purview of a person of skill in the art. Accordingly, in one embodiment, the present invention provides peptides having one of amino acid sequences SEQ ID NOs. 11 to 709 shown in Table 3.

With respect to SEQ ID NO 3, arginine can be replaced with its D-form. As noted above, the D-form of R (r) has been reported to enhance cell penetration ability, something also demonstrated by the present inventors with respect to SEQ ID NO 7. Further, the number of R can be varied from 7 to 9. In this regard, as demonstrated above with respect to SEQ ID NO 2, an siRNA loading region (multiple R region) of 7 R is functional, from which seven or eight continuous R are predicted to work here. Further, the number of glycine, which functions as a spacer can be varied between 0 to 4 (or more, as explained with respect to SEQ ID NO. 2) without significantly affecting the siRNA co-assembly property and transfection capability since this region is not involved in siRNA co-assembly and peptide self-assembly. Accordingly, in one embodiment, the present invention provides peptides having one of amino acid sequences SEQ ID NOs. 710 to 738 shown in Table 4.

The leucine of SEQ ID NO 4 can be replaced with other hydrophobic residues isoleucine, phenylalanine or alanine without altering the helical structure of the original sequence based on secondary structure prediction and accordingly, will have similar siRNA- co-assembly properties. Accordingly, in one embodiment, the present invention provides peptides having one of amino acid sequences SEQ ID NOs. 739 to 741 shown in Table 5.

The leucine of SEQ ID NO 5 can similarly be replaced with other hydrophobic residues isoleucine, phenylalanine or alanine without altering the helical structure of the original sequence based on secondary structure prediction and accordingly, will have similar siRNA-co-assembly properties. Accordingly, in one embodiment, the present invention provides peptides having one of amino acid sequences SEQ ID NOs. 742 to 744 shown in Table 6.

The phenylalanine in SEQ ID NO 6 can suitably be replaced with other hydrophobic residues such as alanine, valine, leucine or isoleucine. Further, the histidine and threonine hydrogen bonding pair can be replaced with other hydrogen bonding pairs, such as H-S, H-N, Q-H, W-H, R-H or Y-H and the position of the members of the hydrogen bonding pairs can be switched. In the self-assembling region, the change of hydrogen bonding pairs and hydrophobic pairs will not significantly alter the siRNA loading region (multiple R region). In addition, these sequences remain amphiphilic in the self-assembling region. Accordingly, these sequences will have similar siRNA loading and transfection abilities as SEQ ID NO 6. Further, the ionic pair R-E can be replaced with R-D or the hydrogen bonding pair of R-H. Accordingly, in one embodiment, the present invention provides peptides having one of amino acid sequences SEQ ID NOs. 745 to 953 shown in Table 7.

With respect to SEQ ID NO 7, the number of glycine, which functions as a spacer can be varied between 0 to 4 (or more, as explained with respect to SEQ. ID. NO 2) without significantly affecting the siRNA co-assembly property and transfection capability since this region is not involved in siRNA co-assembly and peptide self-assembly. Accordingly, in one embodiment, the present invention provides peptides having one of amino acid sequences SEQ ID NOs. 954 to 957 shown in Table 8.

In another aspect, the invention relates to complexes comprising a peptide of the present invention coupled to a cargo molecule by a non-covalent molecular association. While this cargo molecule is not specifically limited, in a preferred embodiment the cargo molecule is a NA and, in a particularly preferred embodiment, siRNA. In one embodiment, the complex comprises a peptide having an amino acid sequence of any one of SEQ ID NOs: 1 to 7. In another embodiment, the complex comprises a peptide having an amino acid sequence of any one of SEQ ID NOs: 8 to 10. In another embodiment, the complex comprises a peptide having an amino acid sequence of any one of SEQ ID NOs: 11 to 709. hi another embodiment, the complex comprises a peptide having an amino acid sequence of any one of SEQ ID NOs: 710 to 738. hi another embodiment, the complex comprises an amino acid sequence of any one of SEQ ID NOs: 739 to 741. In another embodiment, the complex comprises a peptide having an amino acid sequence of any one of SEQ ID NOs: 742 to 744. In another embodiment, the complex comprises a peptide having an amino acid sequence of any one of SEQ ID NOs: 745 to 953. In another embodiment, the complex comprises a peptide having an amino acid sequence of any one of SEQ ID NOs: 954 to 957.

In another aspect the invention relates to compositions comprising a complex or complexes of the present invention. Such compositions can suitably be used as a transfection agent or as a pharmaceutical/medicament.

The advantages of the present invention are further illustrated by the following examples. The examples and their particular details set forth herein are presented for illustration only and should not be construed as a limitation on the claims of the present invention. EXAMPLE 1: LIBRARY AND PEPTIDE DESIGN

Based on the design principles 1 to 7 described below, 45 peptides in 5 categories/groups were designed (Table 9). In this regard, these design principles should not be regarded as limiting on the peptides of the present invention, but only as illustrative of the principles identified as relevant in designing the library described below.

1. Peptides self/co-assembly capability, which can be derived from interaction between amino acids, or amino acids and phosphate groups in siRNA. In International patent application publication no. WO2009/026729, incorporated entirely herein by reference, Chen et al. describe different mechanisms including electrostatic, hydrogen bonding, hydrophobic, and τ-τ stacking interactions incorporated in peptide assembly.

2. Peptide interaction with siRNA through non-covalent interactions such as Columbic forces and hydrogen bonding. In particular, basic amino acids such as lysine and arginine can interact with the negatively charged phosphate groups on the siRNA sugar rings through electrostatic interactions. 3. High siRNA loading capacity so that more siRNA can be delivered at a low cost of materials.

4. Optimal size for cellular internalization through endocytosis, which is about 100 nm to 200 nm. Smaller complexes, depending on their surface properties, can directly penetrate the cell membrane, but can be removed from the blood stream [Moghimi, et al. 1999], Larger particles (>200 nm), on the other hand, can be easily recognized and removed by phagocytic cells [Mahato. 2005]. Furthermore, cellular translocation of larger complexes experiences increasing difficulties. Since endocytosis is one of the most common routes for cellular translocation, the siRNA-peptide complexes should preferably have a size below 200 nm [Owens, et al. 2006]. 5. Surface charge of the siRNA-peptide complexes/assemblies is an essential parameter towards complex aggregation and the triggering of immune response through complement activation. Neutrally charged particles are found with a lower opsonization rate than charged particles due to the decrease in electrostatic interactions with opsonins [Benoit, et al. 2006, Mahato. 2005]. Therefore, the surface charge of siRNA-peptide complexes should preferably be controlled within a narrow range so that the complexes are free from aggregation and have stealth from the immune system.

6. Since various exonucleases are able to degrade siRNA prior to its silencing functioning of protein expression, one of the most important properties of siRNA-carrier complexes/assemblies is to protect the siRNA from premature degradation and other harsh environments, accordingly, the enzymatic and complex stability were considered in library design.

7. The carrier should be biocompatible and not cause any unnecessary side effects. Therefore, a cytotoxicity evaluation of the peptide carriers at effective concentrations has to be carried out.

8. The amino acids used in the peptide sequence design include pH sensitive amino acids, e.g., histidine, as well as basic amino acids, e.g., lysine and arginine.

New Peptide Sequences

Based on the above design principles, 45 peptides in 5 categories/groups were designed (Table 9):

1. The first group mainly consisted of arginine and histidine. Several studies have investigated the translocation efficiency of arginine-rich peptides of various lengths [Langel. 2007, Rothbard, et al. 2002]. It was found that peptides with seven to nine arginine residues have the highest translocation efficiency, while at least five arginine residues are required for translocation to take place. Histidine is a pH sensitive amino acid as it will be protonated at low pH. When a histidine containing peptide is taken in the endosome during endocytosis, it acts as a proton sponge, which disrupts the endosomal pH balance [Akinc, et al. 2005]. This results in the leakage of the endosomal content, releasing the siRNA complexes to the cytosol. The two amino acids, i.e. arginine and histidine, are utilized in the peptide design to combine their cell penetration and endosome disruptive capabilities. The chain length effect of each residue, as well as the effect of charge distribution, is investigated among this group of peptides.

2. The second group was designed to have an α-helical secondary structure having three distinct sections when viewed from the top, each contributed by the amino acids leucine, histidine and arginine. It is believed that hydrophobic residues such as leucine can assist in cell penetration through interacting with the hydrophobic tails in the lipid bilayer, and also assist in pore formation in the cell membrane [Langel. 2007, Li, et al. 2004].

3. Peptide delivery carriers have been designed based on α-helix amphiphilicity while investigations based on amphiphilic /3-sheet peptides are very limited. Therefore, the third group of peptides was designed to explore the possibility of using peptides with /3-strand secondary structures as delivery vehicles [Plenat, et al. 2004, Deshayes, et al. 2004]. This group of peptides consists of complementary amino acid pairing peptides [Chen, et al. 2008], which can self-assemble through complementarity of weak interactions such as electrostatic and hydrophobic forces. The peptides were designed to be sequence complementary as well as possess geometric matches between basic amino acid residues and the phosphate backbone of siRNA.

4. It has been reported that oligo-D-arginine (r) has higher cell penetrating efficiency when compared to the naturally occurring oligo-L-arginine (R) [Wender, et al. 2000]. This group contained some of the peptides of the other groups but with their naturally occurring L- arginine substituted by D-arginine.

5. The final group of peptides contained the derivatives of known peptide sequences such as Tat [Gump, et al. 2007], Penetratin [Dom, et al. 2003], and KLA [Scheller, et al. 2000]. This group incorporates functional moieties of various cell penetrating peptides, endosomal disruptive peptides, and self-assembling peptides. For example, the amino acids lysine and alanine are substituted with arginine and leucine in a KLA peptide.

The transfection efficiency of the siRNA-peptide complexes/assemblies was evaluated in vitro with two cell lines.

Table 9: Peptide library screening results in C166-eGFP cells

M I l I l Highly effective (transfection efficacy of xxx Highly toxic (viability < 20%) positive control Lipofectamine 2000) - Non-toxic (viability > 85%)

- Ineffective (as in untreated cells) MR: Peptide/ siRNA molar ratio

A. A.: Number of amino acids in sequence siRNA concentration: 100 nM

EXAMPLE 2: PEPTIDE-siRNA ASSEMBLIES/NANOPARTICLES PREPARATION IN VITRO AND TRANSFECTION PROCEDURE

Materials and siRNA-peptide Complex/assembly preparation

Two cell lines, used for in vitro experiments, were purchased from the American Type Culture Collection (Rockville, MD, USA). a. Shh-Light II cells (ATCC CRL-2795) were derived from the NIH/3T3 cell line (primary mouse embryonic fibroblast cells) and co-transfected with firefly luciferase reporter and renilla-luciferase expression vector [Sasaki, et al. 1997]. These cells respond to the presence of hedgehog protein by producing light-generating enzymes, which can be measured with a luminometer. The corresponding siRNA, i.e. Anti-Luc siRNA, was purchased from Dharmacon. The molar concentration of siRNA was determined by absorption spectroscopy, using an extinction coefficient of 385103 L/mol cm.

Target sequence: NN GAU UAU GUC CGG UUA UGU A

Sense sequence: GAU UAU GUC CGG UUA UGU A UU Antisense sequence: 5'-U ACA UAA CCG GAC AUA AUC UU b. Mouse endothelial cells C166-GFP (ATCC CRL-2583) were transfected with a plasmid reporter vector, pEGFP-Nl, that encodes the enhanced green fluorescence protein (eGFP) [Wang, et al. 1996]. The corresponding siRNA, i.e., eGFP siRNA, was purchased from Dharmacon with an extinction coefficient of 362408 L/mol cm. Target sequence: GCG ACG UAA ACG GCC ACA AGU Sense sequence: G ACG UAA ACG GCC ACA AGU UC Antisense sequence: ACU UGU GGC CGU UUA CGU CGC

A peptide array consists of crude peptides with N-terminal acetylation and C-terminal amidation was purchased from Pepscan Systems (Leystad, Netherlands). Formulation protocol:

1 hour before transfection, the siRNA solution was prepared in sterile tubes: • Dried siRNA was dissolved in siRNA buffer and vortexed for 10 seconds.

• UV- Vis spectroscopy was used to determine the accurate concentration of siRNA.

30 min before transfection, the siRNA-peptide complex/assembly (and controls) were prepared: • siRNA and peptide solutions were combined in proportion according to the designed experiment and were incubated for 20-30 min at room temperature. siRNA concentrations of 5-100 nM and Peptide/siRNA molar ratios of 10/1 to 50/1 optimized the results in screening experiments.

Depending on their solubility/hydrophobicity, peptides were dissolved in DMSO or Milli-Q water to prepare stock solutions. Peptide and siRNA solutions were diluted in Opti-MEM medium (each in 50 μl)

Transfection protocol

For C166-GFP cell line, 24-well plate and serum-free treatment:

• The cells were seeded with a confluency of 20,000 cells/well in DMEM with 10% FBS without antibacterial agents, 24 hrs before transfection. The confluency of the cells was

40-60% the day of transfection.

• The cells were rinsed with PBS and 150 μL of Opti-MEM, then 100 μL of the complex solution (siRNA-peptide or controls) were added to each well.

• The cells were incubated with the complex at 37°C in a CO2 incubator for 3-6 hours (a period of 4 hours will usually be enough). After incubation, 250 μL DMEM was added with 20% FBS without removing the transfection mixture.

• The cells were fixed in 2% paraformaldehyde (PFA) for the detection of eGFP expression by flow cytometry 24 - 48 hours after the start of transfection:

• Each well was rinsed with PBS and the cells were resuspended by Mg2+ and Ca2+ containing PBS.

• The cells were transferred to centrifuge tubes and centrifuged at 1000 rpm for 10 mins, • The supernatant was removed and the cells were resuspended in 2% PFA for flow cytometry analysis.

For Shh-Light II cell line, 96-well plate and serum-free treatment:

• The cells were seeded with a confluency of 12,000 cells/well in DMEM with 10% FBS without antibacterial agents, 24 hrs before transfection. The confluency of the cells was

40-60% the day of transfection.

• The cells were rinsed with PBS and 20 μL of Opti-MEM was added, then 20 μL of the complex solution (siRNA-peptide or controls) to each well.

• The cells were incubated with the complex at 37°C in a CO2 incubator for 3-6 hours; a period of 4 hours is usually enough. After incubation, 40 μL DMEM was added with 20%

FBS without removing the transfection mixture.

• Dual-Glo luciferase kit was used to measure the activity of firefly and renilla luciferases in treated and control cells:

• 80 μL of Dual-Glo luciferase reagent was added to each well and mix. (A wait of at least 10 minutes is recommended before the next step.)

• The firefly luminescence was then measured using a Luminometer (Floustar Optima, BMG Labtech).

• 80 μL of Dual-Glo Stop & GIo reagent were then added to each well and mix. (A wait of at least 10 minutes is recommended before the next step.) • The renilla luminescence was then measured. The same plate order as the firefly luminescence was measured.

EXAMPLE 3: PEPTmE LIBRARY SCREENING RESULTS

Transfection efficiency of siRNA-peptide formulation in C166-GFP cell line

Flow cytometry is a rapid and sensitive technique for quantifying protein levels in the cell. Fluorescence and light scattering intensity distributions of C166-GFP cells were obtained by flow cytometry (FACS Vantage SE, Becton Dickinson, USA) with a laser excitation wavelength of 488 nm. Fluorescence emission of eGFP is obtained with a 530 ± 30 nm band pass filter. For effective eGFP silencing, the eGFP fluorescence intensity is expected to decrease after siRNA transfection, since the mRNA encoding for the eGFP is degraded. Upon reaching the cytosol, the successfully delivered siRNA would perform RNAi, which prevents the downstream production of eGFP until the siRNA is eventually degraded by endonucleases. However, GFP that is already present in the cytosol prior to siRNA delivery would still give fluorescence before it is degraded by intracellular proteases. Therefore, the effect of silencing was monitored over sufficiently long times (24 hours and 48 hours). The base line of eGFP fluorescence was obtained from the fluorescence of untreated cells, one of the controls. The normal and positive controls were cells transfected with naked siRNA and siRNA-Lipofectamine 2000™ complexes, respectively.

Figures IA-F show the flow cytometry results, indicating fluorescence intensity distributions for the cells treated by some of the promising sequences from the peptide library (Table 9). Fluorescence intensities of the untreated cells and the cells treated by Lipofectamine 2000™ are also shown.

The transfection experiments were performed to screen all sequences in the library.

Table 9 shows the screening results at different peptide/siRNA molar ratios of 10/1, 15/1,

25/1, 40/1, and 50/1. The silencing efficacy of complexes were ranked from (-) ineffective with less than 10% silencing, to (-H-H-++) highly effective with more than 85% silencing of GFP -encoding gene. The cytotoxicity results ranked from (-) nontoxic with more than 85% viability, to ( I I I I I ) highly toxic with less than 20% viability of the cells after treatment.

The results of more accurate cytotoxicity assay (MTT) of the selected sequences are reported in the next section.

As shown in Table 10, the peptides C6, Cl, Bl, A7, A3, E3, and H4 have all shown silencing efficacy with no or limited toxicity. The percentage of silencing in the cells treated by Lipofectamine™ /siRNA and the peptide/siRNA complexes is also shown in Figure 2, for easy comparison. It can be seen that there is no significant difference between the transfection efficacies of the positive control Lipofectamine 2000™ and the peptides Cl and C6. The peptide Bl, which is a combination of ACS [Chen, et al. 2006] and R9 peptides, showed transfection efficiency of 80% of that of Lipofectamine™. The peptides A7, A3, E3, and H4 also demonstrated silencing percentage of higher than 50%. However as demonstrated below, the peptides of the present invention are more biocompatible and less toxic, compared to Lipofectamine 2000™. Figure 2 illlustrates the silencing efficacy of positive control and Peptide-siRNA complexes in C166-GFP cell line. The concentration of the peptides is shown in Table 10.

10

Transfection efficiency of siRNA/peptide formulation in the Shh-Light II cell line

Shh-Light II cells treated by controls and peptide/siRNA complexes/assemblies were analysed for firefly gene activity. The Dual-Glo® luciferase assay system (Promega) was applied according to the manufacture protocol 48 hours post-transfection. This assay provides

15 fast and simple quantitation of a stable luminescent signal from two reporter genes (firefly

SUBSTITUTE SHEET (RULE 26) and renilla luciferases) in a single sample. In this assay, the activity of the primary reporter (firefly) was measured to evaluate the transfection efficacy of the complexes, and the activity of the control reporter (renilla) providing a control to normalize results.

As the luminescent signals are affected by assay conditions, raw results should be converted to normalized ratios. Incorporation of consistent control wells (untreated and treated by Lipofectamine™) on each plate provided the ability to calculate a normalized fϊrefiy/renilla luminescence ratio (F/R) and a Relative Response Ratio (RRR). The RRR can be used to compare results between experiments that do not use the same media/sera combination or have been affected by changes in temperature or other variables. It was defined as:

[_jrjvnteated—[_jrjLipoft ec

According to this equation, the untreated wells and the wells treated by Lipofectamine™ generate RRRs of 1 and 0, respectively.

The activity of renilla luciferase was also used as an indicator of cytotoxicity of the complexes as this luciferase reporter was not affected by Anti-Luc siRNA, and any reduction in the activity level of this reporter after treatment would reflect the toxicity of the complexes.

Figure 3 shows the silencing effect of peptide/siRNA complexes for the promising sequences of the library. It can be seen that peptides Cl and C6 have RRR close to Lipofectamine™ (RRR=O) which indicates high transfection efficacy of these peptides. Other peptides in this Figure had also an RRR of less than 0.5. Considering their low cytotoxicity level in comparison with that of Lipofectamine™ (Figure 4), these peptides are very promising candidates as siRNA delivery carriers.

Figure 3 illustrates the transfection efficacy of promising peptides in comparison with Lipofectamine 2000™ in Shh-Light II cells. See Table 10 for the concentrations of the siRNA and peptides.

SUBSTITUTE SHEET (RULE 26) MTT assay of the selected sequences

To evaluate the viability of the cells treated by complexes, the MTT assay was used for the peptides of high transfection efficacy. All peptide stock solutions were prepared in DMSO and then diluted in Opti-MEM. The toxicity column in Table 9 demonstrates the cytotoxicity of naked peptide solutions in 1-3% DMSO. To eliminate the DMSO effect, the peptide/siRNA complexes were diluted in RNase-free water to obtain a solution of less than 1% DMSO. Figure 4 shows the cytotoxicity of controls, peptides only, and peptide/siRNA complexes against C166-GFP cells. The concentration of peptides corresponded to that of the Peptide/siRNA molar ratio at which the highest transfection efficacy was achieved (Table 10). The viability of the cells treated by the siRNA/peptide complexes is considerably higher than that of the cells treated by Lipofectamine™/siRNA. This indicates higher biocompatibility of the peptides. It is also observed that the complexation did not have significant additional effect on the viability of the cells.

Figure 4 illustrates the cytotoxicity of peptides only and peptides/siRNA complexes against C 166-GFP cells.

To evaluate the toxicity of DMSO, MTT assay was conducted for Peptide A3 in varying concentrations of DMSO (Figure 5). By decreasing the DMSO concentration in the final solution from 2.5% to 0.5%, the viability of the cells was improved by 30%. As most of the peptides in the library are soluble in water or buffer, DMSO should be avoided in the preparation of complexes, due to high cytotoxicity effect. Figure 5 illustrates the DMSO concentration effect on the toxicity of the solution against Cl 66-GFP cells.

Serum effect on the stability and transfection efficacy of the complex

The next screen experiments included serum plus and serum free groups, to investigate the serum effect on the transfection efficacy of the complexes. As mentioned above, the transfection experiments were conducted in the serum-free medium to avoid any effect of serum on the degradation or destabilization of the complex. However, in in vivo transfection, serum is inherently present, so the transfection reagent should be able to preserve and deliver the drug/gene in a serum-plus environment. For this purpose, the complexes of the peptides Cl, C6, and A7 with siRNA were prepared in DMEM with 10% FBS. From Figure 6A, it can be seen that the presence of serum in treatment did not lead to a pronounced reduction in silencing in C166-GFP cells, indicating that the complex was serum stable. siRNA concentration effect on silencing

The results of siRNA transfection experiments reported in Table 9 were performed with 100 nM siRNA. To evaluate the effect of siRNA concentration on transfection efficiency, experiments were conducted with varying siRNA concentrations. As can be seen from Figure 6B, the siRNA concentration of 50 nM seems to be effective in knocking down the gene of interest in C166-eGFP cells. It is important to use the lowest possible concentration of siRNA to avoid potential toxicity and side effects, and decrease the cost of siRNA treatment.

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Claims

1. A complex comprising a peptide and a cargo molecule, wherein the peptide and the cargo molecule are coupled by a non-covalent molecular association.

2. A complex comprising a peptide and a cargo molecule, wherein the peptide has an amino acid sequence selected from the group comprising: n-RLLRLLLRLWRRLLRLLR-c (SEQ. ID. NO 1) n-FQFNFQFNGGGHRRRRRRR-c (SEQ. ID. NO 2) n-ACSSSPSKHCGGGGRRRRRRRRR-c (SEQ. ID. NO 3) n-HRLRHALAHLLHKLKHLLHALAHRLRH-c (SEQ. ID. NO 4) n-LRHLLRHLLRHLRHLLRHLRHLLRHLLRH-c (SEQ. ID. NO 5) n-RFTFHFRFEFTFHFE-c (SEQ. ID. NO 6) n-LAELLAELLAELGGGGrrrrrrrrr-c (SEQ. ID. NO 7).

3. A complex comprising a peptide and a cargo molecule, wherein the peptide has an amino acid sequence of any one of SEQ ID NOs: 8 to 957.

4. A complex according to claim 2 or 3 wherein the cargo molecule is a nucleic acid.

5. A complex according to claim 4 wherein the nucleic acid is a siRNA.

6. A pharmaceutical composition comprising a complex according to claim 5 for delivering a therapeutically effective amount of siRNA.

7. A peptide having an amino acid sequence selected from the group comprising: n-RLLRLLLRLWRRLLRLLR-c (SEQ. ID. NO 1) n-FQFNFQFNGGGHRRRRRRR-c (SEQ. ID. NO 2) n-ACSSSPSKHCGGGGRRRRRRRRR-c (SEQ. ID. NO 3) n-HRLRHALAHLLHKLKHLLHALAHRLRH-c (SEQ. ID. NO 4) n-LRHLLRHLLRHLRHLLRHLRHLLRHLLRH-c (SEQ. ID. NO 5) n-RFTFHFRFEFTFHFE-c (SEQ. ID. NO 6)

n-LAELLAELLAELGGGGrπτrrrrr-c (SEQ. ID. NO 7).

8. A peptide having an amino acid sequence of any one of SEQ ID NOs: 8 to 957.