WO2015084850A1

WO2015084850A1 - Membrane translocation assay for sirna

Info

Publication number: WO2015084850A1
Application number: PCT/US2014/068166
Authority: WO
Inventors: Dennis J. Murphy; Vadim Dudkin; Anthony W. Shaw; Ryan Matthew HAAS; William C. Wimley
Original assignee: Sirna Therapeutics, Inc.
Priority date: 2013-12-03
Filing date: 2014-12-02
Publication date: 2015-06-11
Also published as: WO2015084850A8

Abstract

Disclosed herein is an assay that directly measures the translocation of an siRNA across a lipid bilayer of a liposome. In one embodiment, a process for determining the amount of siRNA translocated across a liposome membrane comprises (a) having a synthetic liposome vesicle with protease chymotrypsin encapsulated inside; (b) having a chymotrypsin inhibitor in bulk solution outside the liposome vesicle; (c) introducing an siRNA to the bulk solution; and (d) measuring the amount of the siRNA in the liposome.

Description

TITLE OF THE INVENTION

MEMBRANE TRANSLOCATION ASSAY FOR SIRNA

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.

61/911,080, filed December 3, 2013, the entire contents of which are incorporated herein by reference.

REFERENCE TO SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCn copy, created on November 26, 2014, is named A2038-7220WO_SL.txt and is 51,259 bytes in size. BACKGROUND OF THE INVENTION

Small interfering RNAs (siRNA) can achieve selective knock-downs of therapeutic targets by degradation of specific messenger RNA, provided the siRNA reaches the RNA Induced Silencing Complex (RISC) in the cell cytosol. Not wishing to be bound by theory, receptor-targeted siRNA constructs are taken up by cell surface receptors and accumulate in subcellular vesicles termed endosomes. A small fraction of the siRNA traverses the endosomal membrane to reach the cytosol. The process, termed endosomal escape, is a major barrier to cytosolic delivery and higher potency of siRNA therapeutics. Having a reliable assay method for measuring this critical event can better enable exploration of the structure activity relationship (S AR) of RNA translocation.

Liposome-based dye leakage assays have been used to study membrane active agents. In these systems a fluorescent-dye/quencher pair is encapsulated in the interior of a liposome. Agents that disrupt the integrity of the membrane cause release of the dye to the bulk solution, resulting in dequenching of the dye and an increase in the observed

fluorescence, e.g., as described in Hassane et al, J. Control Release, 153: 163-172 (2011) and Nir et al, Prog. Lipid Res,. 39: 181-206 (2000). Other variations on liposome based assays include the ability to monitor the life-time of peptide mediated channels (e.g., as described in Krauson et al, ACS Chem. Biol., 8: 823-831 (2013); Krausen et al, Biochim. Biophys. Acta, 1818: 1625-1632 (2012); Krausen et al, /. Am. Chem. Soc, 134: 12732-12741 (2012)) and fusion of individual liposomes with subsequent mixing of the phospholipids making up the membrane bilayer (e.g., as decribed in Thoren et al, Biophys. Chem. , 114: 169-179 (2005)).

The extent of membrane disruption required for siRNA translocation is significantly greater than for low molecular weight dyes (<500 g/mol). It is believed that a much larger channel needs to be created in order for an siRNA with a molecular weight of > 15, 000 g/mol to traverse the membrane. Recent work from Wimley's lab describs an assay for translocating peptides, i.e. peptides that can translocate across membranes without disruption of the bilayer, Wimley et al, ACS Chem. Biol., 5: 905-917 (2010). The system utilizes chymotrypsin and terbium^"1"3 encapsulated within a liposome, and al- AntiTrypsin (al-AT) and lipophilic anion dipicrylamine (DPA) in the bulk solution. Terbium^"1"3 forms a fluorescent chelate with DPA. A disrupting peptide enables Terbium^"1"3 to escape the interior of the liposome and form the fluorescent chelate, allowing the identification of peptides that disrupt the liposome bilayer. See e.g., Marks et al, /. Am.Chem. Soc, 133: 8995 (2011).

Membrane active peptides are capable of facilitating translocation of otherwise impermeable siRNA molecules across biological membranes. These peptides can be combined with siRNA to facilitate endosomal escape. Existing assays for measuring siRNA translocation necessarily introduce the variables of distribution and metabolism, along with RISC loading and mRNA knockdown, to the SAR equation. A direct measure of the effectiveness of peptides in actually shepherding siRNAs across the lipid membrane has been unavailable.

There remains a need for an assay that can accurately and directly measure the level of siRNA translocated across a lipid bilayer of a liposome.

SUMMARY OF THE INVENTION

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Schematic illustration of the principle of siRNA translocation assay.

Figure 2. Results from siRNA translocation assays with melittin and 2 control reactions. Filled circles: siRNA translocation reaction (Example 4); Open squares: lysed liposome control reaction (Example 5); Filled triangles: empty liposome control (Example 6).

Figure 3. Results from siRNA translocation assays with two in vivo active and one in vivo inactive peptides. Filled circles: siRNA Translocation reaction with in vivo active Peptide 1. Filled squares: siRNA Translocation reaction with in vivo active Peptide 2. Filled triangles: siRNA Translocation reaction with in vivo inactive Peptide 3.

Figure 4. Results from siRNA translocation assays with one in vivo active and one in vivo inactive peptide containing targeting ligand. Filled circles: siRNA

Translocation reaction with in vivo active TGN-L-Peptide 4. Open squares: siRNA

Translocation reaction with in vivo inactive TGN-L-Peptide 5. Figure 5. The structure of siRNA- 2 CTNNB1 (1797)_2'-15 SNAP GS.

Figure 6. The structure of siRNA- 3 CTNNB1 (1797) PS05_5' C6.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein is an assay that directly measures the translocation of siRNA across a bilayer liposome membrane, a critical event which is considered to be limiting in vivo potency of siRNA molecules. The instantly disclosed assay measures translocation of siRNA, optionally facilitated by a membrane active peptide, from the exterior to the interior of a liposome due to the transmembrane movement of the siRNA, along with a moiety that selectively generates fluorescence when exposed to the interior of the liposome.

The assay principle is illustrated in Figure 1. Synthetic liposomes are prepared with chymotrypsin encapsulated inside. Chymotrypsin is a digestive enzyme component of pancreatic juice and can hydrolyze amide bonds in peptides. Alpha 1- AntiTrypsin (al-AT), a potent macromolecular inhibitor of chymotrypsin, is added to the bulk solution outside the liposome to quench any residual external enzyme or released enzyme. A fluorescent chymotrypsin sensitive peptide, e.g., ala-ala-phe-aminomethylcoumarin (AAF-amc), is covalently attached to the siRNA to report on siRNA translocation. According to this design, no hydrolysis of the linked AAF-amc occurs in the bulk solution due to inhibition by al-AT. Furthermore non-selective release of chymotrypsin to the bulk solution yields no reaction. Only siRNA that translocates to the interior of an intact liposome is cleaved by chymotrypsin.

Both chymotrypsin and al-AT are macro-molecules (>25,000 g/mol) and should not pass through the liposome membrane.

In one embodiment, a peptide is not necessarily needed as single and double stranded siRNA' s can be directly modified to enable self-transport.

Molecules that cause large scale membrane disruption and breakdown of the liposomes (e.g. liposome lysis) are in general not interesting for therapeutic development due to their non-selective behavior. Control experiments with detergent Triton X-100 showed that lysed liposomes yielded no reaction in the presence of al-AT. Thus the assay disclosed herein does not yield a signal due to liposome lysis. This dramatically reduces the number of uninteresting, detergent-like peptides that would otherwise appear as positives.

Peptides such as melittin are known to cause aggregation and fusion of liposomes at high concentrations. See Wiedman et al, Biochim. Biophys. Acta, 1828: 1357- 1364 (2013). Fused liposomes, due to their larger size, scatter light to a greater extent. Light scattering can result in artificial fluorescence signal. As indicated by examples below, the present siRNA translocation assay can differentiate artificial fluorescent signals from aggregated or fused liposomes and those of the reactions with chymotrypsin.

In addition to the above advantages, the present assay can also have the following additional benefits. First, this assay can model a dual molecular delivery (DMD) approaches in which peptide and siRNA are dosed separately. This enables dissection of pharmacokinetic effects seen in vivo, from true potency in RNA translocation. Variation in the peptide to RNA ratio is achieved as part of the experimental design in which a constant concentration of siRNA is tested against a serial dilution of peptide. One DMD approach is described in patent application having an application number of 61/900542 and the title of Dual Molecular Delivery of Oligonucleotides and Peptide Conjugates, the entire content of which is incorporated herein.

Second, this assay can measure the translocation level of a chemically functionalized siRNA wherein the functionalized siRNA behaves like a translocating peptide and can cross membranes on its own. Cell based or in vivo assays to optimize these molecules suffer from the inability to measure translocation except by mRNA knock-down. A transformative utility of this assay is the ability to explore and optimize the structure-activity relationship of RNA-translocation, unencumbered by the downstream requirements of RISC loading, mRNA binding and cleavage.

In one embodiment, a process for determining the amount of siRNA translocated across a liposome membrane comprises: (a) having a synthetic liposome vesicle with protease chymotrypsin encapsulated inside; (b) having a chymotrypsin inhibitor in bulk solution outside the liposome vesicle; (c) introducing an siRNA to the bulk solution; and (d) measuring the amount of the siRNA in the liposome.

In one embodiment, introduction of the siRNA to the bulk solution in step (c) starts the translocation process of the siRNA across the liposome membrane.

In one embodiment, translocation of an siRNA is started by adding the siRNA to a bulk solution surrounding a synthetic liposome vesicle with chymotrypsin encapsulated inside. In one embodiment, the bulk solution further comprises a membrane active peptide.

In one embodiment, the siRNA is attached to a fluorescent chymotrypsin sensitive peptide. In one embodiment, the fluorescent chymotrypsin sensitive peptide is AAF- amc.

In one embodiment, the AAF-amc is covalently attached to the siRNA through an optional linker.

In one embodiment, the AAF-amc is covalently attached to the siRNA through linker BS(PEG)₅.

In one embodiment, the amount of siRNA in the liposome is measured using a fluorescent assay.

In one embodiment, the liposome membrane comprises lipid components DOPC, DOPE, Soy PI, and DOPG.

In one embodiment, the molar ratio of DOPC, DOPE, Soy PI, and DOPG is 5:2:1:2.

In one embodiment, the chymotrypsin inhibitor in the bulk solution is al-AT.

In one embodiment, the al-AT is present at an amount sufficient to quench the activity of all chymotrypsin in the liposome. In one embodiment, the alphal-AT is present at an amount sufficient to quench the activity of all chymotrypsin, including all chymotrypsin inside the liposome and any residual or leaked chymotrypsin in the bulk solution outside the liposome.

In one embodiment, the translocation of the siRNA from the exterior to the interior of a liposome occurs in the presence of a membrane active peptide, for example, by incubating the siRNA with a membrane active peptide and optionally other reagents in the bulk soution.

In one embodiment, the membrane active peptide is melittin. Melittin is a 26 amino acid membrane active peptide found in bee venom with the sequence

GIGAVLKVLTTGLPALISWIKRKRQQ (peptide SEQ ID NO 8). It can have multiple mechanisms of action including channel formation at low peptide to lipid ratios, and membrane aggregation and fusion at high concentrations.

In one embodiment, the membrane active peptide is selected from peptides having amino acid sequences set forth Table 1.

Table 1. Suitable Peptide Sequences and ID

CGLFGEIHHLIHHGLHNLIDWWNG SEQ ID NO: 26

CGLFGEIHHLIHHGLENLIDWWNG SEQ ID NO: 27

CGLFGEIHELIHHGLENLIDWWNG SEQ ID NO: 28

CGFFGEIAELIEEGLKNLIDWGNG SEQ ID NO: 29

CGLFGEIEELIEEGLENLIDWSNG SEQ ID NO: 30

CGLFGEIEELIEEGLENLIDWPNG SEQ ID NO: 31

CGLFGEIEELIEEGLENLIDWHNG SEQ ID NO: 32

CGLFGEIEELIEEGLENLIDWQNG SEQ ID NO: 33

CGLFGEIEELIEEGLENLIDWENG SEQ ID NO: 34

CGLFEEIAELIEEGLENLIDWGNG SEQ ID NO: 35

CELFEELAELLWEGLENLIDWGNS SEQ ID NO: 36

CGLFGEIAELIWEGLENLIDWGNG SEQ ID NO: 37

CGLLEEIEELLEEGLENLIDWGNS SEQ ID NO: 38

CGLFGEIEELIEEGLENLIDWNNG SEQ ID NO: 39

CGLFAEIEELLEEGLENLIDWGNG SEQ ID NO: 40

CGLFGEIEELIEEGLENLIDWONG SEQ ID NO: 41

CGLFGEIEELIEEGLENLIDWDNG SEQ ID NO: 42

GLFGEIEELIECGLENLIDWGNG SEQ ID NO: 43

CGLFGEIEELIEEGLENLIDW-AIB-NG SEQ ID NO: 44

AC-GLLEEIEELLEEGLENLIDWWNSC SEQ ID NO: 45

GLLEEIEELLEEGLENLAELAEALEALAAGGSC SEQ ID NO: 46

CGLFGEIEELIEEGLENLIDW SEQ ID NO: 47

CGLFGEIEELIEEGLENLID SEQ ID NO: 48

CGLFGEIEELIEEGLENLI SEQ ID NO: 49

CELFEEIAELIEEGLENLIDWG SEQ ID NO: 50

AC-GLFGEIEELIEEGLENLIDWGNGC SEQ ID NO: 51

AC-CGLFGEIEELIEEGLENLIDWGNG SEQ ID NO: 52

AC-CGLFGEIEELIEEGLENLIDWWNG SEQ ID NO: 53

CGFFGEI-AIB-GLLEE-AIB-LHNLIDWWNG SEQ ID NO: 54

CFLGALWKALSELLKNLIDWWNG SEQ ID NO: 55

CGL-R6H-GELEEL-S7H-EEGLENLIDWWNG (STAPLED) SEQ ID NO: 56

CGFFGEI-AIB-ELIWE-AIB-LKNLIDWWNG SEQ ID NO: 57

CGLFEELAGLLWHGLKNLIDWWNG SEQ ID NO: 58

CFLGALFHALSHLLENLIDWWNG SEQ ID NO: 59

CGFF-AIB-EIAELIWE-AIB-LKNLIDWWNG SEQ ID NO: 60

CGLFAEIEELIWEGLENLIDWWNQ SEQ ID NO: 61

STEARYL-AGYLLGKLL-ORN-ORN-LAAAAL-ORN-ORN-LLC SEQ ID NO: 62

R-AHX-R-AHX-RILFQYR-AHX-B ALA-R-AHX-R-B ALA-C SEQ ID NO: 63 R-AHX-RR-B ALA-R-AHX-EIFFQYR-AHX-R-B ALA-R-AHX-

SEQ ID NO: 64

R-B ALA-C

R-AHX-RR-B_ALA-RR-AHX-RILFQYR-AHX-R-B_ALA-R-

SEQ ID NO: 65 AHX-R-B ALA-C

R-AHX-RR-AHX-RR-AHX-RIHILFQNRRMKWHK-B ALA-C SEQ ID NO: 66

CSSAWWSYWPPVA SEQ ID NO: 67

CGLFAVIKKVASVIGGL SEQ ID NO: 68

CGLFAVIHHVASVIGGL SEQ ID NO: 69

CGLFAVIEEVASVIGGL SEQ ID NO: 70

CGPSQPTYPGDDAPVRDLIRFYRDLRRYLNVVTRHRY SEQ ID NO: 71

CGIGAVLHVLTTGLPALISWIKRKRQQ SEQ ID NO: 72

CGIGAVLHVLTTGLPALISWIHHHHQQ SEQ ID NO: 73

AC-GIFEAIAGLLKINFKC SEQ ID NO: 74

AC-GLFGALAEALAEALAEHLAEALAEALEALAAGGSC SEQ ID NO: 75

AC-GLFEAIEGFIENGWEGLAELAEALEALAAGGSC SEQ ID NO: 76

GLFGEIEELIEEGLENLIDWGNGLAELAEALEALAAGGSC SEQ ID NO: 77

GLFEAIEGFIENGWEGLAELAEALEALAAGGSC SEQ ID NO: 78

GLFGALAEALAEALAEHLAEALAEALEALAAGGSC SEQ ID NO: 79

CEENWIGLFGGGNIWEEEEILDLL SEQ ID NO: 80

CGLFGEIEELIEEGLENLIDWGNG SEQ ID NO: 81

CEALFGKINAIFIGKL SEQ ID NO: 82

CGLFGEIEELLEEGLENLIDWGNG SEQ ID NO: 83

CGLFGEIEELIEEALENLIDWGNG SEQ ID NO: 84

CGLFGEIEELIEEGFENLIDWGNG SEQ ID NO: 85

CGLFGEIEELIEEGWENLIDWGNG SEQ ID NO: 86

CGLFGEIEEWIEEGLENLIDWGNG SEQ ID NO: 87

CGLFGEIEEFIEEGLENLIDWGNG SEQ ID NO: 88

CGLFGEIEELFEEGLENLIDWGNG SEQ ID NO: 89

CGLFGEIEELIEEGLENLIDWGNE SEQ ID NO: 90

CGLFGEIEELIEEGLEELIDWGNG SEQ ID NO: 91

CGLFGEIEELIEEGLESLIDWGNG SEQ ID NO: 92

CGLFGEIEELIEEGLEQLIDWGNG SEQ ID NO: 93

CGLFGEIEELIEEGLENWIDWGNG SEQ ID NO: 94

CGLFGEIEELIEEGLENFIDWGNG SEQ ID NO: 95

CGLFGEIEELIEEGLENLWDWGNG SEQ ID NO: 96

CGLFGEIEELIEEGLENLVDWGNG SEQ ID NO: 97

CGLFGEIEELIEEGLENLIEWGNG SEQ ID NO: 98 CGLFGEIEELIEEGLENLIDFGNG SEQ ID NO: 99

CGLFGEIEELIEEGLENLIDLGNG SEQ ID NO: 100

CGLFGEIEELIEEGLENLIDWGYG SEQ ID NO: 101

CGLFGEIEELIEEGLENLIDWGSG SEQ ID NO: 102

CGLFGEI EELI EEGLENLI DWGNQ SEQ ID NO: 103

CGLFGEIEELIEEGLENLIDWGN-AIB SEQ ID NO: 104

CGLFEALLELLESLWELLLEAGYG SEQ ID NO: 105

CGLFEAIEGFIENGWEGMIDWGNG SEQ ID NO: 106

CIFGIDDLEEGLLFVAIVEAGIGGYLLGS SEQ ID NO: 107

CGLFEALLELLESLWELLLEA SEQ ID NO: 108

CGLFGEI EELIEEGLENLIDWGNGC SEQ ID NO: 109

CGNFGEIEELIEEGLENLIDWGNG SEQ ID NO: 121

CGLFAEIEELIEEGLENLIDWGNG SEQ ID NO: 122

CGLFEEIEELIEEGLENLIDWGNG SEQ ID NO: 123

CGLFGEIAELI EEGLENLI DWGNG SEQ ID NO: 124

CELFGEIEELIEEGLENLIDWGNG SEQ ID NO: 125

CALFGEIEELIEEGLENLIDWGNG SEQ ID NO: 126

C-AIB-LFGEIEELIEEGLENLIDWGNG SEQ ID NO: 127

CGWFGEIEELIEEGLENLIDWGNG SEQ ID NO: 128

CGLFGELEELI EEGLENLI DWGNG SEQ ID NO: 129

CGLFGEIEELWEEGLENLIDWGNG SEQ ID NO: 130

CGLFGEIEELIEE-AIB-LENLIDWGNG SEQ ID NO: 110

CGLLGEIEELI EEGLENLI DWGNG SEQ ID NO: 111

CGLFGAIEELI EEGLENLI DWGNG SEQ ID NO: 112

CGFFGEIEELIEEGLENLIDWGNG SEQ ID NO: 113

CGLWGEIEELIEEGLENLIDWGNG SEQ ID NO: 114

CGLFGEWEELIEEGLENLIDWGNG SEQ ID NO: 115

CGLFGEFEELI EEGLENLI DWGNG SEQ ID NO: 116

CGLFGEIEELIEEGLENLLDWGNG SEQ ID NO: 117

CGLFGEI EELIEEGLENLIDWGQG SEQ ID NO: 118

GLFGEIEELIEEGLENLIDWGNG SEQ ID NO: 119

GFFGAIWEFIHSIL SEQ ID NO: 120

In one embodiment, the membrane active peptide is selected from peptides having amino acid sequences as set forth in peptide SEQ ID NO: 3, peptide SEQ ID NO: 4, peptide SEQ ID NO: 6 and peptide SEQ ID NO: 8.

In one embodiment, the membrane active peptide in the bulk solution is further attached to a targeting ligand selected from Table 2.

wherein each n is independently an integer from 1 to 20;

wherein each n is independently an integer from 1 to 20;

wherein n is an integer between 1 and 100;

OH

wherein n is an integer between 1 and 100; and

wherein n is an integer between 1 and 100.

In one embodiment, the membrane active peptide is attached to a targeting ligand through a linker. In one embodiment, the linker is selected from Table 3:

Table 3. Suitable Linkers

n = 0-750. In one embodiment, the membrane active peptide is TGN-L-peptide SEQ ID NO 7, wherein TGN is:

and the linker L is:

In one embodiment, a process for determining the amount of siRNA translocated across a liposome membrane comprises: (a) having a synthetic liposome vesicle with protease chymotrypsin encapsulated inside; (b) having a chymotrypsin inhibitor and a membrane active peptide in bulk solution outside the liposome vesicle; (c) introducing an siRNA with an attached AAF-amc to the bulk solution to start the translocation of the siRNA from the exterior to the interior of the liposome; and (d) measuring the amount of the siRNA in the liposome using a fluorecent assay

In one embodiment, the liposome membrane comprises lipid components DOPC, DOPE, Soy PI, and DOPG. In one embodiment, the molar ratio of DOPC, DOPE, Soy PI, and DOPG is 5:2:1:2.

In one embodiment, the translocation of siRNA occurs in the presence of a membrane active peptide selected from Table 1 , for example, by incubating the siRNA with a membrane active peptide and optionally other reagents.

In one embodiment, the membrane active peptide is further attached to a targeting ligand. In one embodiment, the targeting ligand is selected from Table 2.

In one embodiment, a process for determining the amount of siRNA translocated across a liposome membrane comprises (a) obtaining (i) a synthetic liposome vesicle with a protease chymotrypsin encapsulated inside the liposome vesicle and a bulk solution that comprises a chymotrypsin inhibitor al-AT outside the liposome vesicle; and (ii) an siRNA attached to AAF-amc; (b) introducing the siRNA with the attached AAF-amc and a membrane active peptide to the bulk solution; and (c) measuring the amount of the siRNA in the liposome using a fluorescent assay; wherein the amount of the siRNA in the liposome is a measure of the level of siRNA translocated across the liposome membrane.

EXAMPLES

Examples provided herein are intended to assist in a further understanding of the invention. Particular materials used, species and conditions are intended to be further illustrative of the invention and not limitative of the reasonable scope thereof.

Throughout the application, the following terms have the indicated meanings unless otherwise noted:

Term Meaning

siRNA Small interfering RNA

RISC RNA-induced silencing complex

TON Tetra N- Acetyl Galactosamine targeting ligand

SAR Structure activity relationship

DOPC 1 ,2-Dioleoyl-sn-glycero-3-phosphocholine DOPE l,2-Dioleoyl-sn-glycero-3-phosphatidyl- ethanolamine

DOPG l,2-Dioleoyl-sn-glycero-3-phospho-(l'-rac-glycerol) LBPA Lysobisphosphatidic acid

Soy PI L-a-phosphatidylinositol (Soy) (sodium salt)

DPA Dipicolinic acid

Chymotrypsin 40 U/mg 25 kDalton protease

al-AT al-AntiTrypsin, 44 kDalton protease inhibitor from human plasma

AAF-amc ala-ala-phe-aminomethylcoumarin

Buffer pH 6.0 10 mM MES (2-(N-Morpholino)ethanesulfonic acid, 4-Morpholineethanesulfonic acid), 150 mM NaCl. ICP-AES Inductively coupled plasma atomic emission spectroscopy

LEM Late endosomal membrane

PEG Polyethylene glycol

BS(PEG)5 Bis-succinimide ester-activated PEG compound (with 5- unit polyethylene glycol spacer arms)

The following assay/reaction reagents were used in the following examples and/or procedures: Chymotrypsin: Sigma C4125.

37 U/mg solid

40 U/mg protein (purity unknown)

Mwt = 25 kl)

Stored at 4°C in buffer. al-AntiTrypsin.

Sigma A9024-25MG. alphal-Antitrypsin from human plasma

salt-free, lyophilized powder

Mwt = 44kD

Stored at 4°C in buffer.

AAF-amc; ala-ala-phe-aminomethylcoumarin. Sigma A3401.

Mwt = 464.5 g/mol

10 mM solution in DMSO stored at -20°C.

Triton X-100 reduced. Fluka, Cat #93424, lot 1107805, CAS [92046-34-9]. 10% w/vol solution stored at room temperature

Mellitin from bee venom. Sigma, M7391, 70% (HPLC). 10 mM solution in DMSO.

Empirical Formula: C131H229N39031. Molecular Weight: 2846.46. CAS Number: 20449- 79-0

10 mM solution in DMSO stored at -20oC.

Phospholipids, Avanti:

25 mg/mL chloroform solutions, stored at -20°C.

DOPE Avanti, 850725C,

18:1 (A9-Cis) PE (DOPE), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine

DOPC Avanti, 850357C

18:1 (A9-Cis) PC (DOPC), l,2-dioleoyl-sn-glycero-3-phosphocholine Soy PI Avanti, 840044C

L-a-phosphatidylinositol (Soy) (sodium salt)

DOPG Avanti 840475

18:1 (A9-Cis) PG, l,2-dioleoyl-sn-glycero-3-phospho-(l'-rac-glycerol) (sodium salt) Buffer: 10 mM MES (2-(N-Morpholino)ethanesulfonic acid, 4-Morpholineethanesulfonic acid), 150 mM NaCl.

SIRNA Translocation Assay Procedures siRNA Translocation Assay Procedure - LEM liposomes with encapsulated chymotrypsin were utilized along with al-AT in the bulk solution. The al-AT was at a concentration previously determined to inhibit all of the chymotrypsin in the reaction. The reaction also included a 2X serial dilution of the test peptide. siRNA-1 was added to start the reaction.

In detail, the following reagents were added in order to a Corning 3544 non- binding, low volume, clear bottom 384-well plate:

(1) 1 μΕ of a nine or ten step, 2X serial dilution of peptide in 100% DMSO. The highest concentration of peptide was 50 μΜ final; the lowest was 0.01 or 0.02 μΜ final. A tenth or eleventh reaction was run with DMSO and no peptide;

(2) 30 μΕ of a mixture of 1 mM (final) LEM liposomes containing chymotrypsin and 100 μg/mL (final) of al-AT; and

(3) 9 μΕ of labeled siRNA-1 (26.5 μΜ final).

The plate was covered with foil and spun at 1000 rpm for 1 minute in a table top centrifuge.

The fluorescence of each well due to hydrolysis of the amino-methylcoumarin was recorded on a PerkinElmer Envision fluorometer. (X380 CWL=380nm B W=10nm Tmin=25%, M460 CWL=460nm BW=25nm Tmin=50%; 400 nm dichroic mirror). siRNA Translocation Assay - Lysed Liposome Control Procedure - LEM liposomes with encapsulated chymotrypsin were utilized as above, but lysed with 0.5% Triton X-100. Alphal-AT in the bulk solution was at a concentration previously determined to inhibit all of the chymotrypsin in the reaction. The reaction also included a 2X serial dilution of the test peptide. siRNA- 1 was added to start the reaction.

(1) I μL· oί a nine or ten step, 2X serial dilution of peptide in 100% DMSO. The highest concentration of peptide was 50 μΜ final; the lowest was 0.01 or 0.02 μΜ final. A tenth or eleventh reaction was run with DMSO and no peptide;

(2) 30 μL· of a mixture of 1 mM (final) LEM liposomes containing chymotrypsin, 100 μg/mL (final) of al-antitrypsin, and 0.5% vol/vol (final) of Triton X-100 (reduced); and

(3) 9 μΐ, of labeled siRNA- 1 (26.5 μΜ final).

The plate was covered with foil and spun at 1000 rpm for 1 minute in a table top centrifuge. The fluorescence of each well due to hydrolysis of the amino-methylcoumarin was recorded on a PerkinElmer Envision fluorometer. (X380 CWL=380nm B W=10nm Tmin=25%, M460 CWL=460nm BW=25nm Tmin=50%; 400 nm dichroic mirror). siRNA Translocation Assay - Empty Liposome Control Procedure - LEM liposomes without encapsulated chymotrypsin were utilized along with al-AT in the bulk solution. The reaction also included a 2X serial dilution of the test peptide. siRNA- 1 was added to start the reaction.

(2) 30 μΕ of a mixture of 1 mM (final) empty LEM liposomes, and 100 μg/mL (final) of a 1 -antitrypsin; and

(3) 9 μΐ, of labeled siRNA- 1 (26.5 μΜ final).

The fluorescence of each well due to hydrolysis of the amino-methylcoumarin was recorded on a PerkinElmer Envision fluorometer. (X380 CWL=380nm B W=10nm

Tmin=25%, M460 CWL=460nm BW=25nm Tmin=50%; 400 nm dichroic mirror).

EXAMPLE 1 siRNA Translocation Assay Principal

The translocation assay disclosed herein is depicted in Figure 1. Large- unilamellar liposomes were prepared by extrusion. The phospholipid composition of the liposomes was based on the composition of late endosomal membranes. The enzyme chymotrypsin was encapsulated within the liposomes; the protease inhibitor, al-AT was added to the bulk solution outside the vesicles. To report on translocation a chymotrypsin sensitive peptide (ala-ala-phe-aminomethylcoumarin, AAF-amc) was covalently linked to the siRNA. Cleavage of the amc portion of the peptide by chymotrypsin resulted in an increase in fluorescence. Translocation (movement of the siRNA from the exterior of a liposome to the interior) was facilitated by a membrane active peptide. The al-AT present in the bulk solution quenched the activity of any residual chymotrypsin in bulk solution and enzyme released by extensive membrane lysis. Thus the fluorescence signal was due only to siRNA that translocated to the interior of intact liposomes and cleaved by chymotrypsin.

EXAMPLE 2 Preparation of LEM Liposomes with Chymotrypsin Encapsulated Inside

Liposomes encapsulating chymotrypsin were prepared using a process involving rehydration of a quaternary mixture of phospholipids with a buffer containing chymotrypsin, freeze thawing, extrusion, and purification. The phospholipid composition of the liposomes was: DOPC, DOPE, Soy PI, and DOPG in a molar ratio of 5:2: 1:2.

Specifically, chymotrypsin was encapsulated in LEM liposomes as follows. Chloroform (25 mg/mL) solutions of four phospholipids DOPC (680 μί), DOPE (258 μί), Soy PI (150 μί) and DOPG (276 μί) were combined in a 250 mL round bottom flask and evaporated to dryness on a rotary evaporator, then put under high vacuum for 18 hours. The resulting phospholipids film was re-suspended to 20 mg/mL phospholipids with 1.7 mL of pH

6.0 MES buffer containing 1 mg/mL of chymotrypsin by swirling for 5 minutes. The flask was subjected to 7 freeze/thaw cycles with dry ice with a final return to room temperature. The resulting mixture was pushed through a 0.1 micron membrane with an Avestin Extrusion Apparatus 19 times, in 2 x 1 mL aliquots. An odd number was used so that the final sample had passed through the extrusion membrane. Approximately 1.5 mL of sample was collected.

The sample remained an opaque, milky suspension.

Chymotrypsin was removed from the bulk solution by centrifuging through a 100k Centricon Spin filter (Millipore Amicon Ultra, Ultracel 100k. Regenerated Cellulose. UFC 910024, Lolot R2CA91941). The 1.5 mL sample was diluted up to -10 mL with buffer. The sample was spun at 3000 rcf (relative centrifugal force) for approximately 100 minutes until the volume was less than 2 mL. Two additional rounds of buffer addition and centrifugation were performed to yield approximately 1.5 mL of retentate that was tested for chymotrypsin activity in the bulk solution, and after lysis with 0.5% Triton X-100.

The concentration of phosphorous in the retentate was determined by ICP-AES to be 12.8 mM. Phosphorous concentrations were determined directly by quantitating the amount of phosphorus present in liposomes using an iCAP 6000 Inductively Coupled Plasma (ICP) Spectrophotometer (Thermo Fischer, Pittsburgh, PA). Samples were diluted with water containing 1 ppm Germanium (Ge) (Ricca Chemical Company, Arlington TX) internal standard. After injection, the sample was introduced to Nebulizer source with RF Power 1350 W, Aux gas flow 0.5 L/min and Nebulizer Gas flow 0.65 L/min. Phosphorus content was quantitated using an external standard calibration curve (ranging from 0 ppm to 3 ppm) prepared from NIST Phosphorus (P) ICP standard containing 1 ppm Ge as an internal standard. EXAMPLE 3

Synthesis of Labeled siRNA-1

The labeled siRNA contained a modified duplex siRNA with a covalently attached PEG linker and tripeptide AAF-amc. The compound was synthesized in 3 steps: (1) synthesis of the active PEG-AAF-amc; (2) attachment of the PEG-AAF-amc to a single stranded siRNA; and (3) duplexing with a complementary single stranded siRNA.

Step 1 - Specifically, AAF-amc (6.42 mg, 0.014 mmol) was stirred in 1.5 mL of 1:2 methylene chloride and acetonitrile. BS(PEG)s (21.17 mg, 0.040 mmol) was dissolved in 0.5 mL acetonitrile (0.5 mL), and added to the solution of Ala-Ala-Phe-AMC. The reaction was stirred at room temperature and monitored by TLC and LC/MS. After 1 hour it was loaded directly on a silica gel column and purified on Isco (0-5% MeOH/CH2C12, 12g, 15 min @ 30 mL/min). Fractions containing the compound were dried under vacuum and used without further purification.

Step 2 - siRNA-2 having a sequence of UUUCGAAUCAAUCCAACAG (CTNNBl (1797) 2'-15 SNAP GS; SEQ ID NO: 1) with modifications as shown in Figure 5 (21 mg, 3.03 μιηοΐ) was dissolved in 200 mM NaHC03 pH 8.3 (600 μΐ) and acetonitrile (400 μΐ).

Then a solution of BS(PEG)₅ Ala-Ala-Phe-AMC (200 μΐ, 6.80 μιηοΐ) from the previous step dissolved in acetonitrile (200 uL) was added. After 2 hours the acetonitrile was removed under vacuum and the remaining material diluted with water (2 mL) and purified with an XBridge Prep Phenyl column (5 uM, 19 x 250 mm) using a gradient of 5-45% CH3CN (100 mM TEAA)/ water (100 mM TEA A), 15 min @ 20 mL/min, collect @ 260 nm.

Fractions were dialyzed against water (4 x 15 mL) using a 3K membrane and lyophilized to yield 21.5 mg of CTNNBl (1797) 2'-15 SNAP BS(PEG)₅ Ala-Ala-Phe-AMC GS; 55.5 μΐ,, 0.216 μιηοΐ.

Step 3 - A solution of CTNNBl (1797) 2'-15 SNAP BS(PEG)₅ Ala-Ala-Phe- AMC from the previous step dissolved in water (55.5 uL) was added to siRNA-3 having a sequence of CUGUUGGAUUGAUUCGAAA (CTNNBl (1797) PS05_5' C6; SEQ ID NO: 2) with modifications as shown in Figure 6 (1.6 mg, 0.216 μιηοΐ) in a washed vial.

The solution was heated at 90 °C for 1 minute while vortexing, then cooled to room temperature. Final dilution with 771 uL (~3 mg/mL) yielded siRNA- 1 shown below: siRNA- 1 CTNNBl (1797) PS05_5' C6/ GS (13b5)_ 2'-15 SNAP BS(PEG)₅ Ala- Ala-Phe-AMC

EXAMPLE 4 Peptide Mediated RNA Translocation - Melittin

The bee venom peptide melittin showed dose-dependent translocation of the labeled siRNA-1 with LEM liposomes containing encapsulated chymotrypsin. Figure 2 shows a successful RNA translocation assay in which the fluorescence due to hydrolysis of the labeled siRNA shows a dose dependent increase with melittin, with <2 μΜ melittin showing signal above either control. The 50 μΜ melittin reactions show an increase in signal, likely due to the known ability of melittin to fuse and aggregate liposomes. Translocation of siRNA- 1 across the liposomal membrane occurred due to the membrane active peptide melittin.

Specifically, LEM liposomes containing encapsulated chymotrypsin and al-AT in the bulk solution were incubated with a serial dilution of melittin. The al-AT was at a concentration previously determined to inhibit all of the chymotrypsin in the reaction. The fluorescence of each reaction due to hydrolysis of the amino-methylcoumarin was recorded on a PerkinElmer Envision fluorometer. (X380 CWL=380nm BW=10nm Tmin=25%, M460 CWL=460nm BW=25nm Tmin=50%; 400 nm dichroic mirror).

EXAMPLE 5 siRNA Translocation Assay - Lysed Liposome Control - Melittin

The bee venom peptide melittin showed no reaction with 25 μΜ or lower melittin with labeled siRNA-1 and lysed LEM liposomes in the presence of al-AT sufficient to inhibit all chymotrypsin. LEM liposomes containing encapsulated chymotrypsin were lysed with 0.5% Triton X-100. The Triton X-100 concentration was previously determined to lyse the liposomes and release all of the chymotrypsin to the bulk solution.

Specifically a serial dilution of melittin, lysed liposomes, al-AT and siRNA- 1 were incubated for 3 hours. The fluorescence of each reaction due to hydrolysis of the amino- methylcoumarin was also recorded on a PerkinElmer Envision fluorometer. (X380

CWL=380nm BW=10nm Tmin=25%, M460 CWL=460nm BW=25nm Tmin=50%; 400 nm dichroic mirror).

The results of this lysed liposome control assay are also shown in Figure 2.

EXAMPLE 6 siRNA Translocation Assay - Empty Liposome Control - Melittin

A control reaction was run with liposomes lacking chymotrypsin at concentrations of melittin known to cause liposome aggregation and fusion. As shown in Figure 2, the bee venom peptide melittin showed fluorescence signal due to light scattering with 50 μΜ melittin with labeled siRNA-1, empty LEM liposomes, and al-AT. This indicates that the signal in the Lysed Liposome Control at 50 μΜ melittin (Example 5) is likely due to light scattering and not hydrolysis of the labeled siRNA-1. These results suggest that the siRNA translocation assay disclosed herein works as designed.

Specifically a serial dilution of melittin, empty liposomes, al-AT and siRNA-1 were incubated for 3 hours. The fluorescence of each reaction was recorded on a PerkinElmer Envision fluorometer. (X380 CWL=380nm BW=10nm Tmin=25%, M460 CWL=460nm

BW=25nm Tmin=50%; 400 nm dichroic mirror).

EXAMPLE 7 Peptide Mediated RNA Translocation - Active and Sequence Scrambled Analogs

Two in vivo active peptide analogs showed dose-dependent translocation of the labeled siRNA- 1 with LEM liposomes containing encapsulated chymotrypsin, whereas an in vivo inactive peptide analog did not show translocation. The results are shown in Figure 3.

Specifically, LEM liposomes containing encapsulated chymotrypsin and al-AT in the bulk solution were incubated with a serial dilution of Peptide 1

(cglfgeieelieeglenlidwwng; SEQ ID NO 3), Peptide 2 (cglfgeieelieeglenlidwgng; SEQ ID NO 4) and Peptide 3 (ceenwiglfgggniweeeeildll; SEQ ID NO 5), respectively. The al-AT was at a concentration previously determined to inhibit all of the chymotrypsin in the reaction.

The fluorescence of each reaction due to hydrolysis of the amino- methylcoumarin was recorded on a PerkinElmer Envision fluorometer. (X380 CWL=380nm

BW=10nm Tmin=25%, M460 CWL=460nm BW=25nm Tmin=50%; 400 nm dichroic mirror). The slope of the reaction for the first 2 hours was used to determine translocation.

Suitable peptides/conjugates include: EXAMPLE 8

Peptide Mediated RNA Translocation - Active and Sequence Scrambled Analogs Containing Targeting Ligand

An in vivo active peptide analog with covalently attached TON targeting ligand showed dose-dependent translocation of the labeled siRNA- 1 with LEM liposomes containing encapsulated chymotrypsin, whereas an in vivo inactive TGN-peptide analog did not show translocation. The results are shown in Figure 4.

Specifically, LEM liposomes containing encapsulated chymotrypsin and al-AT in the bulk solution were incubated with a serial dilution of TGN-L-Peptide 4 (TGN-L- cifgaiagfikniwegli; SEQ ID NO: 6) and TGN-L-Peptide 5 (TGN-L-cealfgkinaifigkl; SEQ ID

NO: 7), respectively, wherein TGN-L- is:

The al-AT was at a concentration previously determined to inhibit all of the chymotrypsin in the reaction. The fluorescence of each reaction due to hydrolysis of the amino- methylcoumarin was recorded on a PerkinElmer Envision fluorometer. (X380 CWL=380nm BW=10nm Tmin=25%, M460 CWL=460nm BW=25nm Tmin=50%; 400 nm dichroic mirror).

The slope of the reaction for the first 2 hours was used to determine translocation.

One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The methods and compositions described herein, as presently representative of preferred embodiments, are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art, which are encompassed within the spirit of the invention, are defined by the scope of the claims.

Claims

WHAT IS CLAIMED IS:

1. A process for determining the amount of siRNA translocated across a liposome membrane, comprising:

(a) having a synthetic liposome vesicle with protease chymotrypsin encapsulated inside;

(b) having a chymotrypsin inhibitor in bulk solution outside the liposome vesicle;

(c) introducing an siRNA to the bulk solution; and

(d) measuring the amount of the siRNA in the liposome.

2. The process of claim 1, wherein introduction of the siRNA to the bulk solution in step (c) starts the translocation of the siRNA across the liposome membrane.

3. The process of claim 1, wherein the siRNA is attached to a fluorescent chymotrypsin sensitive peptide.

4. The process of claim 3, wherein the fluorescent chymotrypsin sensitive peptide is AAF-amc.

5. The process of claim 4, wherein the AAF-amc is covalently attached to the siRNA through an optional linker.

6. The process of claim 5, wherein the AAF-amc is covalently attached to the siRNA through linker BS(PEG)₅.

7. The process of claim 5, wherein the amount of siRNA in the liposome is measured using a fluorescent assay.

8. The process of claim 1, wherein the liposome membrane comprises lipid components DOPC, DOPE, Soy PI, and DOPG.

9. The process of claim 8, wherein the molar ratio of DOPC, DOPE, Soy PI, and DOPG is 5:2: 1:2.

10. The process of claim 1, wherein the chymotrypsin inhibitor in the bulk solution is al-AT.

11. The process of claim 10, wherein the al-AT is present at an amount sufficient to quench the activity of all chymotrypsin in the liposome.

12. The process of claim 1, wherein the siRNA is translocated across the liposome membrane in the presence of a membrane active peptide selected from Table 1.

13. The process of claim 12, wherein the membrane active peptide is selected from peptides having amino acid sequences as set forth in peptide SEQ ID NO: 3, peptide SEQ ID NO: 4, peptide SEQ ID NO: 6 and peptide SEQ ID NO: 8.

14. The process of claim 13, wherein the membrane active peptide is further attached to a targeting ligand selected from Table 2.

15. The process of claim 14, wherein the membrane active peptide is attached to the targeting ligand through a linker selected from Table 3.

16. The process of claim 1, comprising:

(b) having a chymotrypsin inhibitor and a membrane active peptide in bulk solution outside the liposome vesicle;

(c) introducing an siRNA with an attached AAF-amc to the bulk solution to start the translocation of the siRNA from the exterior to the interior of the liposome; and

(d) measuring the amount of the siRNA in the liposome using a fluorecent assay.

17. The process of claim 16, wherein the liposome membrane comprises lipid components DOPC, DOPE, Soy PI, and DOPG.

18. The process of claim 17, wherein the molar ratio of DOPC, DOPE, Soy PI, and DOPG is 5:2: 1:2.

19. The process of claim 16, wherein the membrane active peptide is selected from peptides having amino acid sequences as set forth in peptide SEQ ID NO: 3, peptide SEQ ID NO: 4, peptide SEQ ID NO: 6 and peptide SEQ ID NO: 8.

20. A process for determining the amount of siRNA translocated across a liposome membrane, comprising:

(a) obtaining:

(i) a synthetic liposome vesicle with a protease chymotrypsin encapsulated inside the liposome vesicle and a bulk solution that comprises a chymotrypsin inhibitor al-AT outside the liposome vesicle;

(ii) an siRNA attached to AAF-amc; (b) introducing the siRNA with the attached AAF-amc and a membrane active peptide to the bulk solution; and

(c) measuring the amount of the siRNA in the liposome using a fluorescent assay; wherein the amount of the siRNA in the liposome is a measure of the level of siRNA translocated across the liposome membrane.