# Patent

## Title
Peptide sequence design and use thereof for peptide-mediated siRNA delivery

## Abstract

The invention provides, in one aspect, peptides and a complex comprising one of the peptides and a cargo molecule, wherein the peptide and the cargo molecule are coupled by non-covalently. The peptides of the invention were found to facilitate the delivery of siRNA molecules into cells and to function in siRNA mediated silencing of cellular targets.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a 35 USC §371 national stage application of PCT Application No. PCT/CA2012/050843, filed Nov. 23, 2012; which claims priority under the Paris Convention to U.S. Application No. 61/563,591, filed on Nov. 24, 2011. The entire contents of the above-referenced applications are expressly incorporated herein by reference.


FIELD OF THE INVENTION
The present invention relates to peptide sequences and their use for short interfering RNA (siRNA) delivery.
BACKGROUND OF THE INVENTION
Over the past decade, we have witnessed tremendous progress in our understanding of the role of RNA molecules in the regulation of gene expression. The main contribution to this progress was offered by the discovery of RNA interference (RNAi). First identified in C. elegans by Fire and Mello [Fire, et al. 1998], RNAi is an evolutionary conserved mechanism that brings about a sequence specific, post transcriptional gene silencing (PTGS) through the use of short RNAs. The basic idea behind RNAi is that a short RNA duplex of 21-23 nucleotides, termed short interfering RNA or siRNA, complementary to a segment of the mRNA, can be exogenously synthesized and introduced into the cell. This triggers a process which finally degrades the homologous mRNA and inhibits the production of the corresponding protein. Several types of short RNAs, including short interfering RNA (siRNA), micro RNA (miRNA), tiny non-coding RNA (tncRNA), and short hairpin RNA (shRNA), may be involved in RNAi process [Novina, et al. 2004, Paddison, et al. 2008, Engelke, et al. 2005].
The major limitations for the use of siRNA both in vitro and in vivo are the instability of naked siRNA in physiological conditions, rapid clearance from the bloodstream, and the inability to cross the cellular membrane to gain access to the intracellular environment. Because of their small size and hydrophilicity, a significant portion of administrated naked siRNAs are excreted through the reticuloendothelial system (RES) [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, nucleic acid (NA)-based drugs are subjected to enzymatic degradation during circulation and within the cell, resulting in insufficient drug potency at the target site. Chemical modifications in the sugars, nucleobases, and the phosphate ester backbone of siRNA have been applied to improve its nuclease resistance without interfering with the silencing efficiency [Manoharan 2004, Verma, et al. 2003, Zhang, et al. 2006]. Conjugation with hydrophobic functional groups has also enhanced the cellular uptake [Oliviera et al., 2006].
In comparison with chemical modifications of NAs, which is time-consuming and costly, carrier-mediated strategies are emerging as a simple and fast means to formulate NA therapeutics and protect them from degradation. The carriers, including viral vectors, lipids, polymers, and peptides, co-assembled or covalently conjugated with siRNA, are designed to enhance cell targeting, prolong drug circulation time, and improve membrane permeation.
Because of their diversity and versatility in design, through the use of amino acids with different physicochemical properties, peptides have been employed to deliver synthetic drugs, small molecules, bioactive peptides, therapeutic proteins, and NAs by a mechanism that has not yet been fully understood. These peptides may include protein-derived cell penetrating peptides (CPPs) [Langel 2007], cationic peptides [Benoit, et al. 2006], designed amphiphilic peptides [Oehlke, et al. 1998], fusogenic peptides [Mok, et al. 2008], cell targeting peptides (CTP) [vives 2005], and peptides containing a nuclear localization signal [Cartier, et al. 2002]. Cationic peptides rich in basic amino acids can electrostatically interact with small NAs or condense NA into small stable particles. CPPs can facilitate the translocation of the complex through the cell membrane. Histidine-rich pH-sensitive or fusogenic peptides can enhance the endosomal escape and cytoplasmic release of the gene complex. Involvement of CTPs in gene delivery systems mediates cell and/or tissue-specific targeting. Finally, attachment of a NLS peptide improves nuclear localization of the gene complex.
Among CPPs, only few have shown high transfection efficiency with low cytotoxicity and immunogenicity. Tat and Penetratin are the most widely investigated peptides among protein-derived peptides. Trans-activating transcriptional activator (Tat) from Human Immunodeficiency Virus 1 (HIV-1), discovered by Frankel and Pabo in 1988, can be efficiently taken up by several cell types in culture [Jervert, et al. 2007].
Specific cell-penetrating peptides (CPPs) identified as effective 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. U.S. Pat. No. 7,163,695 to Mixson and U.S. Pat. No. 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. U.S. Pat. No. 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. U.S. Pat. No. 7,306,784 to Piwinica-Worms describes use of cell membrane-permeant peptide conjugate coordination and covalent complexes having target cell specificity.
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.
International patent application publication no. WO2010/039088 to Kariem et al. describes the use of some stearylated linear or branched CPPs, in particular Transportan and Penetratin, in NA delivery.
U.S. Pat. No. 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. 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.
SUMMARY OF THE INVENTION
In one embodiment, there is provided a peptide having the amino acid sequence selected from SEQ. ID. NO 1 to SEQ. ID. NO 46.
In another embodiment, the present invention provides a complex of a peptide and a cargo molecule, wherein the peptide has the amino acid sequence selected from SEQ. ID. NO 1 to SEQ. ID. NO 46.
In a preferred embodiment, the cargo molecules of the present invention are NAs and, in a particularly preferred embodiment, the cargo is siRNA.
In another embodiment, there is provided a pharmaceutical composition of the complex for delivering a therapeutically effective amount of siRNA.
In another embodiment, there is provided a method for reducing the levels of a gene product within a cell or tissue of an animal comprising administering a therapeutically effective amount of siRNA.
In another embodiment, there is provided a method for reducing the levels of a gene product that regulates apoptosis.



BRIEF DESCRIPTION OF THE DRAWINGS
This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
FIG. 1 illustrates the helical wheel projections of C6M and C6M1 peptides.
FIG. 2 illustrates intracellular silencing of GAPDH gene in CHO cells.
FIG. 3 illustrates silencing efficiency of peptides-siRNA complex, with two Molar Ratios: 20 and 40. FIGS. 3A and 3B show two different runs.
FIG. 4 illustrates the cytotoxicity of peptide-siRNA complexes against CHO-K1 cells. FIG. 4A shows 18 peptide-siRNA complexes. FIG. 4B shows 6 peptide-siRNA complexes.
FIG. 5 illustrates the size of the C6M1-siRNA complex in PBS over time.
FIG. 6 illustrates fluorescence spectroscopy results of C6M1-siRNA complexes at different molar ratios with a fixed concentration of C6M1.
FIG. 7 illustrates fluorescence spectroscopy results of C6M1 and labelled siRNA complexes at different molar ratios with a fixed concentration of C6M1.
FIG. 8 illustrates in vivo results. FIG. 8A shows photographs of tumor tissue where mice were killed by cervical dislocation before tumors were separated. FIG. 8B shows antitumor activity of siRNA/C6M1 complex in a mouse tumor model; wherein the complexes were administered intra-tumorly in mice model bearing A549 cancer cells xenografted under the skin and tumor sizes were measured everyday. FIG. 8C shows body weights of the mice, measured everyday during treatment.



DETAILED DESCRIPTION OF PREFERRED EMBODIMENT
The peptide-cargo complexes of the present invention are formed by a non-covalent molecular association through weak interactions between peptide and siRNA, which provides a simple and fast means to formulate siRNA therapeutics.
The molecular association of the complexes of the present invention is 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.
Considering the amphiphilic nature of the cell membrane, certain designed peptides possess both hydrophilic and hydrophobic moieties. The hydrophilic side interacts with hydrophilic drugs/genes, and the hydrophilic heads of the lipid bilayer through electrostatic interaction, while the hydrophobic side is anchored in the hydrophobic core of the bilayer, assisting the translocation of peptide-cargo to the cytosol.
I. C1 Peptides:
In one embodiment, the present invention provides peptides having the following amino acid sequences:







TABLE 1







C1 family peptide sequences









Name
Sequence
Seq. ID number





C1 M
FQFNFQFNGGGPKKKRKV
(SEQ. ID. NO 1)


 


C1 M1
FQFNFQFNGGGPKPKRKV
(SEQ. ID. NO 2)


 


C1 M2
FQFNFQFNFQFNGGGPKKKRKV
(SEQ. ID. NO 3)


 


C1 M3
FQFNFQFNFQFNWSQPKPKRKV
(SEQ. ID. NO 4)


 


C1 M4
FQFNFQFNFQFNGGGPKPKRKV
(SEQ. ID. NO 5)


 


C1 M5
FQFNFQFNFQFNGGGCHHRRRRRRHC
(SEQ. ID. NO 6)


 


C1 M6
FQFNFQFNFQFNGGGCPKPKRKVC
(SEQ. ID. NO 7)









This group of peptides was designed to explore the possibility of using peptides with β-strand secondary structures as delivery vehicles. The hydrophobic and hydrophilic segments at two opposite ends of these sequences are joined through a linker. In international patent application publication no. WO2009/026729, Chen et al. describe different mechanisms including electrostatic, hydrogen bonding, hydrophobic, and π-π stacking interactions incorporated in peptide assembly. These strategies were applied in designing the hydrophobic section of this class of peptides which can self assemble through complementarity of weak interaction such as hydrogen bonding and hydrophobic forces. The length of hydrophobic section has also changed in C1M2 to C1M4 from 8 to 12 amino acids, to evaluate the significance of hydrophobic segment length.
The linker, GGG, was used to link the hydrophobic and hydrophilic together while keeping the flexibility in the peptide backbone. The linker was changed in C1M3 to WSQP to evaluate any possible effect of linker [Deshayes, et al. 2004].
The positively-charged hydrophilic segment is responsible for both co-assembling with siRNA and approaching the cell membrane through electrostatic interaction between guanidino groups in argenine and lysine of the peptide and phosphate groups in siRNA backbone and phospholipid bilayers of the cells. A nuclear localization sequence (NLS), PKKKRKV, in C1M replaced the argenine-rich segment of peptide C1, with a sequence of FQFNFQFNGGGHRRRRRRR, originally reported in international patent application publication no. WO2011/020188 to Chen et al. (the entire disclosure of which is incorporated herein by reference). The sequence was further modified by a single mutation of a lysine residue in NLS to a proline residue in order to limit its nuclear translocation and rapid release of the cargo in the cytoplasm in C1M1, C1M3, and C1M4. The hydrophilic segment was also replaced with CHHRRRRRRHC and CPKPKRKVC in C1M5 and C1M6, respectively, to evaluate the effect of cystine amino acid in enhancing the release of siRNA from the complex due to the pH change in endosome and cytoplasm.
II. E3 Peptides:
In another embodiment, the present invention provides peptides having the following amino acid sequences:







TABLE 2







E3 family peptide sequences









Name
Sequence
Seq. ID number





E3M
RLTLHLRLELTLHLE
(SEQ. ID. NO 8)


 


E3M1
RWTWHWRWEWTWHWE
(SEQ. ID. NO 9)









This group of peptides was also designed based on amino acid paring strategies mentioned by Chen et al. in international patent application publication no. WO2009/026729. However, in this class of peptides, unlike C1 family peptides, the hydrophilic amino acids, R and H, are distributed among the hydrophobic residues. The peptide E3, with a sequence of RFTFHFRFEFTFHFE, was originally reported in international patent application publication no. WO2011/020188 by Chen et al. Here, we have further modified this sequence. The self-assembly of these sequences is favored by electrostatic interaction between R and E, hydrogen bonding between T and H, and hydrophobic interaction between F's in E3, L's in E3M, and W's in E3M1. The histidine residues were also used to enhance the endosomal escape for the complex as it is protonated at low pH. When a histidine containing peptide is taken into the endosome during endocytosis, it may act as a proton sponge, disrupting the endosomal pH balance, releasing the complex from the endosome. The hydrophobic amino acids, L and W in E3M and E3M1, respectively, replaced F residues in E3 to evaluate the effect of hydrophobic strength of peptides. Furthermore, tryptophan residues were reported to facilitate translocation of the peptide through the cell membrane.
III. A7 Peptides:
In another embodiment, the present invention provides peptides having the following amino acid sequences:







TABLE 3





A7 family peptide sequences



















A7M
RHALAHLLHKLKHLLHALAHR
(SEQ. ID. NO 10)



 



A7M1
RHALAHLLHRLRHLLHALAHR
(SEQ. ID. NO 11)










The group, mainly consisted of arginine, lysine, histidine, leucine, and alanine, 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 and/or alanine, arginine and/or lysine, and histidine. It is believed that hydrophobic residues such as leucine may assist in cell penetration through interacting with the hydrophobic tails in the lipid bilayer of the cell, and also assist in pore formation in the cell membrane [Langel 2007]. Several studies have investigated the translocation efficiency of arginine-rich peptides of various lengths. It was found that peptides with seven to nine arginine residues have the highest translocation efficiency, while at least five arginine/lysine residues are required for translocation to take place. Histidine, as mentioned above, is a pH sensitive amino acid as it will be protonated at low pH. When a histidine containing peptide is taken into the endosome during endocytosis, it may act as a proton sponge, disrupting the endosomal pH balance. This may result in the leakage of the endosomal content, releasing the siRNA complexes to the cytosol. Peptide A7, with a sequence of HRLRHALAHLLHKLKHLLHALAHRLRH, originally reported in international patent application publication no. WO2011/020188 by Chen et al. Here, we evaluated the sequence length effect, as well as the effect of replacement of argenine with lysine in A7M and A7M1.
IV. C6 Peptides:
In another embodiment, the present invention provides peptides having the following amino acid sequences:







TABLE 4







C6 family peptide sequences









Name
Sequence
Seq. ID number





C6M
RLWRLWLRLWRRLWRLLR
(SEQ. ID. NO 12)


 


C6M1
RLWRLLWRLWRRLWRLLR
(SEQ. ID. NO 13)


 


C6M2
RLWRLLWHLWRHLWRLLR
(SEQ. ID. NO 14)


 


C6M3
RLWHLLWRLWRRLHRLLR
(SEQ. ID. NO 15)


 


C6M4
HLLRLLLRLWHRLWRLLR
(SEQ. ID. NO 16)


 


C6M5
HLWHLLLRLWRRLLRLLR
(SEQ. ID. NO 17)


 


C6M6
GLWHLLLHLWRRLLRLLR
(SEQ. ID. NO 18)


 


C6M7
GLWHLLLHLWRRHHRHHR
(SEQ. ID. NO 19)


 


C6M8
GLWHLHLHLWRRHHRLLR
(SEQ. ID. NO 20)


 


C6M9
GLWHLLLHLWHRLLRHHR
(SEQ. ID. NO 21)









This group of peptides was designed based on peptide C6, reported by Chen et al, in international patent application publication no. WO2009/026729 (the entire disclosure of which is incorporated herein by reference). C6 was designed to contain seven positively charged arginine residues. Arginine rich peptides are reported to be capable of delivering siRNA into cells with high efficiency and low toxicity [Futaki, et al., 2001; Wang, et al., 2006] while at least five arginine residues are required to maintain the transfection efficiency. Hydrophobic residues such as leucine are also included in C6 sequence. These hydrophobic amino acids can facilitate the translocation of peptide by interacting with the hydrophobic tails in the lipid bilayer or assisting in pore formation in the cell membrane [Langel. 2007]. It has been found that alanine, leucine and histidine are found abundantly in the helical regions of proteins [Chou and Fasman 1973]. They indicate that leucine is the strongest structure forming residue in the proteins they investigated.
C6 sequence was modified to increase its solubility and transfection efficiency, and series of derivatives were obtained. The derivatives were designed to allow positioning of hydrophilic and hydrophobic residues on opposite faces of the molecule, upon adapting a helical structure. FIG. 1 shows the amino acids have been predicted to position the nonpolar and polar amino acids in the two different faces of the helix, possibly to facilitate self/co assembly. Some hydrophobic leucine residues are selectively substituted with less hydrophobic tryptophan residues to improve their solubility in water. Meanwhile, the aromatic tryptophan is reported to play an essential role in the cellular uptake of many cell-penetrating peptides [Derossi, et al. 1994; Heitz, et al. 2004], as they are able to interact with the lipid/cholesterol within the cell membrane. Histidine is a pH sensitive amino acid, suitable to be contained in a peptide sequence exhibiting the ability to interact with siRNA molecules and to favor the escape of siRNA from endosomes, due to the protonation effect of its imidazole ring at pH 6 [Lo & S. Wang, 2008]. This results in the leakage of the endosomal content, releasing siRNA into the cytoplasm where RNA interference happens. Tryptophan and histidine, were added at different positions in the peptide sequence to combine their cell penetration and endosomal disruption capabilities. The C6 derivatives incorporate functional moieties of various co-assembling peptides, cell penetrating peptides and endosomal disruptive peptides.
V. MW Peptides:
In another embodiment, the present invention provides peptides having the following amino acid sequences:







TABLE 5







MW family peptide sequences









Name
Sequence
Seq. ID number





MW1
MWKSKIGSWILVRWAMWSKKRPKP
(SEQ. ID. NO 22)


 


MW2
MWKSHIGSWILVRWAMWSHKRPKP
(SEQ. ID. NO 23)


 


MW3
MWKSKISWILVSKPGLCKKRPKP
(SEQ. ID. NO 24)


 


MW4
MHKSKISWHLVSKPGLCHKRPKP
(SEQ. ID. NO 25)









This group of peptides is derived from N-terminal sequence (1-30) of the bovine prion protein (bPrPp). The bovine PrP with the sequence of MVKSKIGSWILVLFVAMWSDV GLCKKRPKP adopts a largely a-helical structure [Biverståhl, et al. 2004]. The basic sequence of bovine PrP (residues 23-28) resembles a nuclear localization sequence (NLS) and stimulates cellular uptake by lipid raft-dependent and macropinocytosis [Wadia, et al. 2008]. The transfection efficiency of Bovine PrP is reported to be 48% [Endoh & Ohtsuki, 2009]. Thus, some trypotophan and histidine residues are added to the sequence to increase the affinity of the peptide with membrane and promote endosomal escape.
VI. D-Form Peptides:
In another embodiment, the present invention provides peptides having the following amino acid sequences:







TABLE 6







D-form peptide sequences











Name
Sequences
Seq. ID number






C6-Dr
rLLrLLLrLWrrLLrLLr
(SEQ. ID. NO 26)


 



C6-D
rllrlllrlwrrllrllr
(SEQ. ID. NO 27)


 



C6M1-Dr
rLWrLLWrLWrrLWrLLr
(SEQ. ID. NO 28)


 



C6M1-D
rlwrllwrlwrrlwrllr
(SEQ. ID. NO 29)


 



C6M3-Dr
rLWHLLWrLWrrLHrLLr
(SEQ. ID. NO 30)


 



C6M3-D
rlwhllwrlwrrlhrllr
(SEQ. ID. NO 31)


 



C6M6-Dr
GLWHLLLHLWrrLLrLLr
(SEQ. ID. NO 32)


 



C6M6-D
glwhlllhlwrrllrllr
(SEQ. ID. NO 33)









For the peptide mediated drug delivery system, the peptide is not only responsible for the delivery of siRNA, but also plays an important role in protecting siRNA from the degradation by nucleases in serum, which will influence the gene silencing efficiency. This gives us the hint to make modifications of some peptide sequences.
One of the obstacles in transfection is the cell membrane. It has been reported that D-form of R(r) could enhance the cell penetration ability and enhance the serum stability. The L-form of R was replaced with the D-form of R(r) in sequences of C6, C6M1, C6M3 and C6M6.
Using the D-form of R(r) also affects the secondary structure of the peptide, and correspondingly may influence the shape and size of the peptide-siRNA complex. Peptide sequences C6, C6M1, C6M3 and C6M6 were converted into total D-form to alter transfection efficiency, stability, and cytotoxicity.
VII. GL Peptides:
In another embodiment, the present invention provides peptides having the following amino acid sequences:







TABLE 7







GL peptide sequences









Name
Sequence
Seq. ID number





GL1
GLWRAWLWKAFLASNWRRLLRLLR
(SEQ. ID. NO 34)


 


GL2
GLWRASWLKAWLASNWHKKHRLLR
(SEQ. ID. NO 35)


 


GL3
GLWGAWFIEGWEGMIDGRRLLRLLR
(SEQ. ID. NO 36)


 


GL4
GLWRASWLKAFLASNWHKKLHKK
(SEQ. ID. NO 37)


 


HA2-C6
GLFGAIAGFIENGWEGMIDGRLLR
(SEQ. ID. NO 38)



LLLRLWRRLLRLLR



 


HA2-PK
GLFGAIAGFIENGWEGMIDGWYGPK
(SEQ. ID. NO 39)



KKRKV









This group comprises additional W and H amino acids which may increase the affinity of peptides with cell membranes and increase endosomal escape. Also the n-terminal domain was replaced with GLW, which is reported to be important in CADY [Karidia, et al. 2009] and MPG [Simeoni, et al. 2003] peptides.
HA2, GLFGAIAGFIENGWEGMIDGWYG, has been added to facilitate the endosomal escape for some CPPs, e.g., Penetratin and HA2-Penetratin, and was proved to increase the transfection efficiency of penetratin. Moreover, an added nuclear localization sequence (NLS), PKKKRKV with lysine-rich segments, has shown high nuclear and cytoplasmic localization properties [Simeoni, et al. 2003].
VIII: Stearic Acid (STR) Modified Peptides
In another embodiment, the present invention provides peptides having the following amino acid sequences:







TABLE 8







STR modified peptide sequences









Name
Sequence
Seq. ID number





STR-C1
CH3(CH2)16-GGGPKPKRKV
(SEQ. ID. NO 40)


 


STR-HK
CH3(CH2)16-HHHPKPKRKV
(SEQ. ID. NO 41)


 


STR-HKC
CH3(CH2)16-HHHCPKKKRKVC
(SEQ. ID. NO 42)









Stearic acid is one of the fatty acids occurring abundantly in the body and has high interaction ability with the cell membrane. It has been reported that the cell penetrating peptides with stearic acid modification at the N-terminal could increase the cellular uptake of siRNA-peptide complex [Tanaka, et al. 2010], which may be due to the increased affinity to the cell membrane mediated by stearic acid. Meanwhile, it is believed that stearic acid modified PEI could increase siRNA protection from degradation in Fetal Bovine Serum (FBS) as compared to the parent PEI [Alshamsan, et al. 2009].
In view of these reports, stearic acid was incorporated into the C1 family peptides to increase the membrane affinity of the hydrophobic domain and to enhance siRNA stability in serum. As in C1 family peptides the hydrophobic segments are separated from hydrophilic parts, it is easier to replace the hydrophobic domain with stearic acid to evaluate the effect of different hydrophobic segments. Three peptides were chosen from C1 family with different degree of hydrophobic interaction with cell membrane, and modified with stearic acid.
Meanwhile, the linker GGG was changed in STR-HK and STR-HKC to HHH to enhance the endosomal escape of the complex, as it is protonated at low pH and also to evaluate the effect of a different linker.
IX. Cysteamide (CYSt) Modified Peptides
In another embodiment, the present invention provides peptides having one of the following amino acid sequences:







TABLE 9







CYSt modified peptide sequences









Name
Sequence
Seq. ID number





C6-CYSt
RLLRLLLRLWRRLLRLLR-Cysteamide
(SEQ. ID. NO 43)


 


C6M1-CYSt
RLWRLLWRLWRRLWRLLR-Cysteamide
(SEQ. ID. NO 44)


 


C6M3-CYSt
RLWHLLWRLWRRLHRLLR-Cysteamide
(SEQ. ID. NO 45)


 


C6M6-CYSt
GLWHLLLHLWRRLLRLLR-Cysteamide
(SEQ. ID. NO 46)









This group of peptides was modified with cysteamide group at the C-terminal. Cysteamide is the simplest stable aminothiol and a degradation product of amino acid cysteine. It has been reported that attaching cysteamide group at the C-terminus of peptide could promote efficient transfection of DNA-peptide complexes [Simeoni, et al. 2003]. It has also been studied that peptides modified with cysteamide at C-terminus shows higher potency to cross cell membranes than the original peptide [Crombez, et al. 2009].
This hypothesis has been extended to siRNA-peptide delivery system. To investigate the effect of cysteamide group in enhancing the transfection efficiency of complexes, peptides from C6 family with different levels of transfection efficiency were chosen and modified with cysteamide group at C-terminal.
Example 1
Peptide-siRNA Assemblies/Nanoparticles: Preparation and Transfection In Vitro
The ability of promising peptides to deliver siRNA was evaluated on CHO cells using GAPDH as the target gene. Chinese hamster ovary cells CHO-K1 (ATCC CCL-61) were derived as a subclone from the parental CHO cell line initiated from a biopsy of an ovary of an adult Chinese hamster. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a housekeeping gene widely expressed in diverse tissues and cell types and functions in a variety of cellular processes. The corresponding siRNA, i.e. Silencer® GAPDH siRNA (Human, Mouse, Rat), was purchased from Ambion. The molar concentration of siRNA was determined by absorption spectroscopy, using an extinction coefficient of 385103 L/mol cm.
A peptide array consisting of crude peptides with N-terminal acetylation and C-terminal amidation was purchased from Pepscan Systems (Leystad, Netherlands).
Formulation Protocol:
siRNA concentrations of 50 nM and Peptide/siRNA molar ratios of 20/1 and 40/1 were used in screening experiments.
Transfection Protocol
The cells were seeded with a confluency of 35,000 cells/well in F12K medium with 10% FBS without antibacterial agents, 24 hrs before transfection.
On the next day, rinse the cells with PBS buffer before transfection and add 200 μL of Opti-MEM. 100 μL of the complex solution (negative control siRNA-peptide and GAPDH siRNA-peptide) were added to each well.
The cells were incubated with the complex at 37° C. in an incubator. After 4 hours, 300 μL F12-K with 20% FBS was added without removing the transfection mixture.
The cells were rinsed and lysed 48 hours after the start of transfection.
Real-time RT-PCR is at present the most sensitive method for the detection of low abundance mRNA. Total RNA from the cells was extracted with TRIzol reagent (Invitrogen, Carlsbad, Calif., USA), then treated with chloroform (Sigma, Oakville, Ontario, Canada) and 2-propanol (Sigma, Oakville, Ontario, Canada) as recommended by the manufacturer. The RNA concentrations were measured by Nanodrop (Nanodrop spectrophotometer ND-1000, Thermo scientific, Ottawa, Canada). All the RNAs were reverse transcribed with Bio-Rad iScript cDNA synthesis kit according to the protocol. The cDNA synthesis was primed with a unique blend of oligo (dT) and random primers. PCR was performed with primers for mouse GAPDH gene with: 5′-TTGCTGTTGAAGTCGCAGGAG-3′ (Primer 1, SEQ ID NO: 47) and 5′-TGTGTCCGTCGTGGATCTGA-3′ (Primer 2, SEQ ID NO: 48) (Sigma, Oakville, Ontario, Canada). To avoid bias, real-time RT-PCR is usually referred to one or several internal control genes, which should not fluctuate during treatments [Nicot, et al. 2005]. Here, the house keeping gene cyclophilin was chosen as an internal control to normalize GAPDH gene. Normalization was performed by the amplification of mouse/rat cyclophilin mRNA with the following primers 5′-AGGGTTTCTCCACTTCGATCTTGC-3′ (Primer 3, SEQ ID NO: 49) and 5′-AGATGGCACAGGAGGAAAGAGC AT-3′ (Primer 4, SEQ ID NO: 50) (Sigma, Oakville, Ontario, Canada).
RT-PCR results of GAPDH siRNA complexed with different peptides at molar ratio 20 and 40 are shown in FIG. 2. FIG. 2 shows the intracellular silencing of GAPDH gene in CHO cells where siRNA were complexed with peptides at a molar ratio of 20:1 and 40:1. mRNA levels were measured 48 hours after transfection. The results shown in FIG. 2 correspond to an average of at least three separate experiments.
Example 2
Formulation Protocol can Refer to the Above Example
Transfection Protocol
For CHO-K1 cell line, 96-well plate and serum-free treatment:
The cells were seeded with a confluency of 5000 cells/well in F12K medium 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 50 uL of Opti-MEM was added, then 50 uL of the complex solution (siRNA-peptide or controls) was 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 is usually enough. After incubation, 50 uL F12K medium with 30% FBS was added without removing the transfection mixture.
The cells were rinsed and lysed 48 hours after the start of transfection.
GAPDH KDalert kit was used to measure the activity of enzymatic activity of glyceralde-hyde-3-phosphate dehydrogenase (GAPDH) in treated and control cells:
The KDalertTM GAPDH Assay Kit is a rapid, convenient, fluorescence-based method for measuring the enzymatic activity of glyceralde-hyde-3-phosphate dehydrogenase (GAPDH) in cultured cells derived from human, mouse, and rat. The KDalert GAPDH Assay Kit is designed to facilitate identification of optimal siRNA delivery conditions by assessment of GAPDH expression and knockdown at the protein level.
GAPDH is a tetrameric enzyme, composed of 36 kD protein subunits. It catalyzes the oxidative phosphorylation of glyceraldehyde-3-phosphate (G-3-P) to bisphosphoglycerate (BPG):
The KDalert GAPDH Assay measures the conversion of NAD+ to NADH by GAPDH in the presence of phosphate and G-3-P. Under the recommended assay conditions, the rate of NADH production is proportional to the amount of GAPDH enzyme present. Thus the assay can be used to accurately determine the amount of GAPDH protein in a sample.
Experimental Results:
FIG. 3A shows the silencing efficiency of peptides-siRNA complex, with two Molar Ratio: 20 and 40. FIG. 3B shows the results of a different run for the silencing efficiency of peptides-siRNA complex, with the same Molar Ratios. FIG. 4A shows the cytotoxicity of 18 peptide-siRNA complexes against CHO-K1 cells. FIG. 4B shows the cytotoxicity of 6 peptide-siRNA complexes against CHO-K1 cells.
Example 3
Size of the Peptide-siRNA Complexes
Dynamic Light Scattering (DLS) method was applied to measure the size of the complex in Zeta Sizer Nano Series (Malvern). FIG. 5 shows the size of the complexes of C6M1 peptide and siRNA at peptide/siRNA molar ratios from 1:1 to 60:1 over time. As shown, the size of the complex increases over time, by increasing peptide/siRNA molar ratio. As the size range of 100 to 250 nm might be the appropriate range for in vitro and in vivo treatment, the molar ratio of 20:1 with an incubation time of less than 30 min might be the optimum conditions for complex preparation.
Example 4
Fluorescence Spectroscopy
The peptide C6M1 was used in this experiment to study the interaction between peptide and siRNA using fluorescence spectroscopy method. C6M1 has four tryptophan amino acids which can be used as fluorescence probe. As shown in FIG. 6, by adding more siRNA to samples of fixed C6M1 concentration, the intensity of fluorescence decreased, indicating the interaction between peptide and siRNA. A blue shift from 355 to 330 nm was also observed in molar ratios of less than 40:1, clearly at 20:1 and 10:1, probably due to the change in the environment of tryptophan by adding more hydrophilic siRNA molecules.
A similar experiment was also performed by tracking the fluorescence property of labeled siRNA, interacting with C6M1. As shown in FIG. 7, no significant change in the fluoresce spectra of siRNA was observed at low molar ratios of 2:1 and 4:1. However by adding more peptides, the intensity decreased at molar ratios of 10:1 and 20:1, indicating the interaction and enclosing the siRNA by peptide molecules. The red shift could also be an indicator of a change in the siRNA environment and/or conformational change in siRNA structure upon interacting with peptides.
Example 5
C6M1/siRNA Complex Inhibits Proliferation of Cancer Cells in Nude Mice
The potency of C6M1/siRNA complexes to inhibit cancer cell proliferation was investigated on a tumor animal model. siRNA targeting the bcl-2 gene product was used here. The bcl-2 protein regulates the mitochondria-mediated apoptosis pathway, and various cell death stimuli, including chemotherapeutic agents. So that a drug to reduce the levels of this protein would be expected to promote apoptosis and would therefore be considered a promising therapeutic agent.
The animal model was established by subcutaneous inoculation of 5×106 A549 cells in BALB/c nude mice at the right armpit. When the tumor volume reached 100-200 mm3, the complexes were injected. The C6M1/siRNA complexes were prepared as described before and injected directly into the tumor. Treatments were given every 3 days, for a total of nine treatments at the dose of 4 μg siRNA per mouse. The body weight and tumor diameters were measured everyday. The tumor volume was calculated as follows:

Tumor volume=0.5×(width)2×length.

Mice were killed on the 27th day after first injection. Results corresponding to the average of two different animals.

The results are shown in FIG. 8. These results suggested that bcl-2 siRNA complexed with peptide C6M1 specifically inhibits tumor growth, as a significant reduction in the tumor size was observed after the treatment. Moreover, the complexes showed low toxic effect. During treatment, if the average weight of mice in treated group (tumor tissue exclusive) have decreased (self-control) by more than 15%, it indicates that the drug has toxicity reaction, then the dosage should be reduced to re-test. In our experiment, the body weight of six mice in each group did not change significantly during the treatment. All these results demonstrate that C6M1 is an effective and safe tool for in vivo siRNA delivery.
Mice were killed by dislocation, separate tumor and weighed it. We calculated average weight of each group and tumor inhibition rate. The siRNA group inhibition rate is 37%, siRNA-C6M1 53%, which can inhibit tumor growth significantly.
Although the invention has been described with reference to certain specific embodiments, various modifications thereof will be apparent to those skilled in the art without departing from the purpose and scope of the invention as outlined in the claims appended hereto. Any examples provided herein are included solely for the purpose of illustrating the invention and are not intended to limit the invention in any way. Any drawings provided herein are solely for the purpose of illustrating various aspects of the invention and are not intended to be drawn to scale or to limit the invention in any way. The disclosures of all prior art recited herein are incorporated herein by reference in their entirety.
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## Claims

1. A complex comprising a peptide having the amino acid sequence according to any one of SEQ ID NOs: 12 to 21, 28 to 33, and 44 to 46 and a cargo molecule.

2. The complex according to claim 1, wherein the cargo molecule is a nucleic acid.

3. The complex according to claim 2, wherein the nucleic acid is short interfering RNA (siRNA).

4. The complex of claim 3, wherein the siRNA is complexed with the peptide at a molar ratio within the range of 1:1 to 60:1.

5. The complex of claim 3, wherein the siRNA is complexed with the peptide at a molar ratio within the range of 5:1 to 60:1.

6. The complex of claim 3, wherein the siRNA is complexed with the peptide at a molar ratio of 20:1.

7. The complex of claim 3, wherein the siRNA is complexed with the peptide at a molar ratio of 40:1.

8. A pharmaceutical composition comprising a complex according to claim 3 for delivering a therapeutically effective amount of siRNA.

9. A method of reducing the levels of a gene product within a cell or tissue of an animal comprising administering the pharmaceutical composition of claim 8.

10. The method of claim 9, wherein the cell is a tumor cell or the tissue is tumor tissue.

11. The method of claim 9, wherein the siRNA targets a gene product that regulates apoptosis.

12. The method of claim 11, wherein the gene that regulates apoptosis is bcl-2.

13. A peptide having the amino acid sequence according to any one of SEQ ID NOs: 12 to 21, 28 to 33, and 44 to 46.

14. The pharmaceutical composition of claim 8, wherein the siRNA is complexed with the peptide at a molar ratio within the range of 1:1 to 60:1.

15. The method of claim 9, wherein the siRNA is complexed with the peptide at a molar ratio within the range of 1:1 to 60:1.

16. A method of delivering siRNA into a cell, comprising administering the complex of claim 3 to the cell.

17. The method of claim 16, wherein the cell is a CHO cell.

18. The method of claim 16, wherein the siRNA reduces the levels of an endogenous protein of the cell.

19. The method of claim 16, wherein the siRNA is complexed with the peptide at a molar ratio within the range of 1:1 to 60:1.