WO2011011631A2

WO2011011631A2 - Nucleic acid delivery vehicles

Info

Publication number: WO2011011631A2
Application number: PCT/US2010/042951
Authority: WO
Inventors: Samuel Zalipsky; Li Wang; Zhongli Ding; Bing Luo
Original assignee: Samuel Zalipsky; Li Wang; Zhongli Ding; Bing Luo
Priority date: 2009-07-22
Filing date: 2010-07-22
Publication date: 2011-01-27
Also published as: WO2011011631A3

Abstract

The invention provides a stable nanoparticle for nucleic acid delivery to cells and tissues in vitro and in vivo, methods for making and using the nanoparticle for at least therapeutic and diagnostic purposes.

Description

Nucleic Acid Delivery Vehicles

Field of the Invention

[0001] The invention relates to chemistry,

biochemistry and molecular biology.

Background of the Invention

[0002] Nucleic acids are used in a growing number of therapeutic applications. Therapeutic nucleic acids' effectiveness is related to their delivery to and activity in cells and tissues. Thus, nucleic acid delivery involves bundling and maintaining a nucleic acid in a vehicle for transport through the body and entry into a cell of a sufficient amount of nucleic acid and of sufficient activity to be effective. Such vehicles desirably have a small size to permit

transport to the desired sites and entry into target cells and maintain such size in compositions such as formulations for administration and during transport in vivo in serum. Further, a nucleic acid delivery system must be non-toxic or of low toxicity. Some therapies also require that the vehicle have the ability to release the nucleic acid within the target cell or tissue . [0003] Delivery of nucleic acids such as small interference RNA (siRNA) remains a major obstacle for therapeutic applications. Accordingly, there is an urgent need for a nucleic acid delivery vehicle that effectively delivers a nucleic acid into a target cell such that the nucleic acid had the desired activity in that cell.

Summary of the Invention

[0004] The invention provides a stable nucleic acid delivery vehicle in the form of a nucleic acid- containing nanoparticle, also referred to herein as a nanoplex (NPX) , capable of systemic delivery of nucleic acids, with demonstrated ability to deliver functional nucleic acids into a cell in vitro and in vivo. A NPX of the invention has one or more desirable properties including low toxicity, biodegradability, a mean diameter of 40-150 nm or less that is stable under various conditions including salt, serum and storage, resistance to RNAse mediated degradation of the nucleic acid, reduced leakage of the nucleic acid from the NPX, reduced interaction with polymorphonuclear (PMN) cells and peripheral blood mononuclear cells (PBMC) ,

internalization into a target cell in vitro and in vivo and in embodiments comprising an antisense nucleic acid, ability to specifically knock down of target molecule expression. Nucleic acids delivered by a NPX of the invention are distributed within the cell outside the endosome and have functional activity in the cell, such as the ability to reduce the expression or activity of a target molecule in the cell.

[0005] The invention further provides compositions, including pharmaceutical compositions comprising a NPX of the invention, methods for making the NPX and methods of using the NPX to deliver nucleic acids, such as therapeutic nucleic acids to a target cell or tissue.

[0006] Particular non-limiting embodiments of the invention are set forth in the following numbered paragraphs .

1. A nanoparticle comprising a nucleic acid and a composition formed by combining a conjugate of a cationic polymer covalently linked to a hydrophilic polymer with unconjugated cationic polymer in a molar ratio of conjugated cationic polymer: unconjugated cationic polymer of at least 1:19, wherein the nucleic acid and the cationic polymer in said composition form a non-covalent complex, and wherein said composition comprises a biodegradable cross-linker, said

nanoparticle having two or more properties selected from the group consisting of:

a) a mean diameter of less than 150 nm;

b) a mean diameter of less than 150 nm in 100 mM NaCl for at least 30 min;

c) a mean diameter of less than 150 nm after 30- fold concentration;

d) retains the nucleic acid in serum; and

e) internalizes in a target cell.

2. The nanoparticle of paragraph 1, wherein the nucleic acid is selected from the group consisting of: DNA, LNA, RNA, DNA-RNA hybrids, PNA.

3. The nanoparticle of paragraph 1, wherein the nucleic acid is RNA and is selected from the group consisting of: mRNA, miRNA, tRNA, tmRNA, rRNA and

StRNA.

4. The nanoparticle of paragraph 1, wherein the nucleic acid is an antisense nucleic acid.

5. The nanoparticle of paragraph 4, wherein the antisense nucleic acid is selected from the group consisting of: siRNA, shRNA, miRNA, ribozymes .

6. The nanoparticle of paragraph 1, wherein the nucleic acid comprises a modified backbone linkage selected from the group consisting of: phosphorothioate linkages, phosphoramidate linkages, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates, phosphinates, phosphoramidates and aminoalkylphosphoramidates , thionophosphoramidates , thionoalkylphosphonates , thionoalkylphosphotriesters , morpholino linkages; siloxane linkages; sulfide, sulfoxide and sulfone linkages; formacetyl and

thioformacetyl linkages; methylene formacetyl and thioformacetyl linkages; alkene linkages; sulfamate linkages; methyleneimino and methylenehydrazino

linkages; sulfonate and sulfonamide linkages and amide linkages.

7. The nanoparticle of paragraph 1, wherein the nucleic acid comprises a modified nucleotide selected from the group consisting of: a purine derivative, a pyrimidine derivative, non-natural nucleotides, nucleotides comprising modifications such as 2'-O- methyl, 2'-0' alkyl, 2'-S-alkyl, 2'-fluoro-, 2' -halo, OMe (2' -0-methyl) , F (2 ' -fluoro) , ANA (altritol nucleic acid), HNA (hexitol nucleic acid), AEM (2'- aminoethoxymethyl) and APM (2 ' -aminopropoxymethyl) HM (4' -C-hydroxymethyl-DNA) , ADA (2'-N- adamantylmethylcarbonyl-20-amino-LNA (locked nucleic acid)) PYR (2 ' -N-pyren-l-ylmethyl-2 ' -amino-LNA) , EA (2' -aminoethyl) , GE (2 ' -guanidinoethyl) , CE (2'- cyanoethyl) , AP (2 ' -aminopropyl) , OX (oxetane-LNA) [, CLNA (2' , 4' -carbocyclic-LNA-locked nucleic acid), CENA (2' , 4' -carbocyclic-ENA-locked nucleic acid) ,AENA (2'- deoxy-2' -N, 4' -C-ethylene-LNA) .

8. The nanoparticle of paragraph 1, wherein the nucleic acid is single stranded. 9. The nanoparticle of paragraph 1, wherein the nucleic acid is double stranded.

10. The nanoparticle of paragraph 1, wherein the nucleic acid comprises:

a) an overhang of 1-4 nucleotides;

b) two strands each with an overhang of 1-4 nucleotides;

c) a blunt end; or

d) two strands with blunt ends.

11. The nanoparticle of paragraph 1, wherein the nucleic acid is an antisense nucleic acid having a length of 15-30 nucleotides. 12. The nanoparticle of paragraph 1, wherein the nucleic acid is an antisense nucleic acid having a length of 19-30 nucleotides. 13. The nanoparticle of paragraph 1, wherein the nucleic acid is an antisense nucleic acid having a length of 25 nucleotides. 14. The nanoparticle of paragraph 11, wherein the nucleic acid is an antisense nucleic acid and wherein the nanoparticle internalizes in a cell and reduces expression of a target molecule by at least 10%. 15. The nanoparticle of paragraph 14, wherein the expression of a target molecule is reduced by at least 50 %.

16. The nanoparticle of paragraph 1, wherein the nucleic acid is siRNA.

17. The nanoparticle of paragraph 1, wherein the nucleic acid is 25 base pair, blunt-ended double stranded siRNA.

18. The nanoparticle of paragraph 1, wherein the hydrophilic polymer is selected from the group

consisting of: polyethlyene glycol (PEG), poly (2'- methyloxyline) , poly (N-vinylpyrolidone) ,

poly (hydroxypropyl methacrylate) , poly (2-hydroxymethyl methacrylate) and polyacrylamide .

19. The nanoparticle of paragraph 18, wherein the hydrophilic polymer is PEG and wherein the PEG is selected from the group consisting of linear PEG, branched PEG and dendrimeric PEG. 20. The nanoparticle of paragraph 19, wherein the PEG is selected from the group consisting of:

monofunctional PEG, homobifunctional PEG and

heterobifunctional PEG.

21. The nanoparticle of paragraph 19, wherein the PEG is an alkylating or acylating PEG.

22. The nanoparticle of paragraph 19, wherein the PEG has a formula weight selected from the group consisting of: 2 KD, 3.5 KD, 5 KD 8 KD and 10 KD.

23. The nanoparticle of paragraph 1, wherein the hydrophilic polymer is covalently joined to the

cationic polymer via bond selected from the group consisting of: amide, urethane, ether, secondary amine, thioether, disulfide, ester, acetal, orthoester, ketal and enzyme-cleavable bonds. 24. The nanoparticle of paragraph 1, wherein the covalent bond is stable under physiological conditions.

25. The nanoparticle of paragraph 1, wherein the cationic polymer is selected from the group consisting of: polyethyleneimine (PEI), a co-polymer of histidine and a non-histidine amino acid that has a positive charge at a physiological pH, chitosan analogs,

polylysine, polyornithine . 26. The nanoparticle of paragraph 24, wherein the cationic polymer is a co-polymer of histidine and a non-histidine amino acid that has a positive charge at a physiological pH and is selected from the group consisting of: a linear co-polymer, a branched co^¬ polymer, a dendrimeric co-polymer.

27. The nanoparticle of paragraph 1, wherein the cationic polymer is a polymer selected from the group consisting of:

a) CKHHH-KHHH-KHHH-KHHHKC (SEQ ID NO: 1 ) ;

b) (KHKHHKHHKHHKHHKHHKHK)₂K;

c) (CKHKHHKHHKHHKHHKHHKHK)₂K;

d) (KHHH-KHHH-KHHH-KHHHK)₄KKK;

e) KKK(KHHH-KHHH-KHHH-KHHHK) _4;

f) (CKHHH-KHHH-KHHH-KHHHK)₄KKK;

g) (KHHH-KHHH-KHHHH-KHHHK)₄KKK;

h) (KHHH-KHHHH-KHHHH-KHHHK) ₄KKK;

i) KKK(KHHH-KHHHH-KHHHH-KHHHK)₄;

j) (KHHH-KHHH-KHHH-KHHHK)₈KKKKKKK;

k) any one of a)-j) in which one or more lysines are replaced by an amino acid selected from the group consisting of: 2 , 5-diaminopentanoic acid, 2,4- diaminobutanoic acid and 2, 3-diaminopropionic acid; and 1) any one of a)-j) in which any of the core lysines is replaced by arginine, norarginine or ornithine .

28. The nanoparticle of paragraph 1, wherein the cationic polymer in said hydrophilic polymer-cationic polymer conjugate and said unconjugated cationic polymer are different cationic polymers. 29. The nanoparticle of paragraph 1, wherein said composition is formed by combining said hydrophilic polymer-cationic polymer conjugate with said

unconjugated cationic polymer in a molar ratio of at least 1:19.

30. The nanoparticle of paragraph 1, wherein said cross-linking agent is selected from the group

consisting of: Dimethyl 3 , 3 " -dithiobispropionimidate (DTBP), Dithiobis (succinimidylpropionate) (DSP) and 3 , 3 ' -dithiobis (sulfosuccinimidylpropionate) (DTSSP) .

31. The nanoparticle of paragraph 1, wherein the mean diameter is 100 nm or less.

32. The nanoparticle of paragraph 16, comprising two or more two different siRNAs, wherein the different siRNAs are selected from the group consisting of:

a) siRNAs that target a different sequence in the same target nucleic acid;

b) siRNA's that target different target nucleic acids,- and

c) siRNAs with and without a chemical

modification.

33. The nanoparticle of paragraph 1, wherein the nucleic acid is siRNA, the hydrophilic polymer is mPEG, the cationic polymer is HK copolymer PT- 91, wherein said composition comprises:

a) a conjugate of mPEG covalently bonded with an amine of HK copolymer PT- 91, wherein the ratio of mPEG molecules to HK copolymer PT- 91 molecules is 1:1; and b) unconjugated HK copolymer PT-91.

34. The nanoparticle of paragraph 33, wherein said composition further comprises cross-linking.

35. The nanoparticle of paragraph 33, wherein said composition is formed by combining the mPEG-PT-91 conjugate with unconjugated PT-91 in a molar ratio of at least 1:19.

36. The nanoparticle of paragraph 35, wherein the mPEG is selected from the group consisting of: mPEG 2K, mPEG 3.4K, mPEG 5K, mPEG 8K and mPEG 1OK. 37. The nanoparticle of paragraph 33, produced by a method comprising the steps of

a) combining a conjugate of mPEG covalently linked to an amine of a PT-91 compound, wherein the ratio of mPEG molecules to PT-91 molecules is 1:1, with

unconjugated PT-91 to form a mixture of conjugated and unconjugated PT-91;

b) combining the mixture produced in step a) with said siRNA such that the siRNA and the PT-91 form a non-covalent complex, thereby producing a nanoparticle.

38. The nanoparticle of paragraph 33, wherein in step a) , the conjugated and unconjugated PT-91 are combined at a molar ratio of at least 1:19. 39. The nanoparticle of paragraph 33, further comprising the step: c) contacting the nanoparticle produced in step b) with Dimethyl 3 , 3 " -dithiobispropionimidate 2 HCl to produce a cross-linked nanoparticle. 40. The nanoparticle of paragraph 37, wherein the nanoparticle produced in step b) is contacted with 50, 75 or 100 molar excess of dithiobispropionimidate 2 HCl to PT-91. 41. The nanoparticle of paragraph 1 or paragraph 33, wherein the nucleic acid is detectably labeled.

42. The nanoparticle of paragraph 35, wherein the siRNA is detectably labeled.

43. The nanoparticle of paragraph 1, paragraph 32 or paragraph 35, further comprising one or more

components selected from the group consisting of: a fusogenic molecule, a targeting moiety and an endosomal lysing agent.

44. A composition comprising a nanoparticle of paragraph 1, paragraph 33 or paragraph 35 and a

pharmaceutically acceptable carrier.

45. The composition of paragraph 42, further comprising an additional active component which is a therapeutic agent. 46. A kit comprising a container, the

nanoparticle of paragraph 1, paragraph 33 or paragraph 35 or the composition of paragraph 44 and instructions for use. 47. A method for making the nanoparticle of paragraph 1, comprising the steps of:

a) combining a conjugate of a hydrophilic polymer covalently linked to a cationic polymer with

unconjugated cationic polymer to form a mixture,- b) combining the mixture produced in step a) with said nucleic acid such that the nucleic acid and the cationic polymer form a non-covalent complex, thereby producing a nanoparticle; and

c) contacting the nanoparticle produced in step b) with a cross-linking agent to produce a cross-linked nanoparticle . 48. A method for making the nanoparticle of paragraph 33, comprising the steps of:

a) combining a conjugate of mPEG covalently bonded with an amine of PT- 91, wherein the ratio of mPEG molecules to PT- 91 molecules is 1:1, with unconjugated PT- 91 to form a mixture,- and

b) combining the mixture produced in step a) with siRNA such that the nucleic acid and the PT- 91 form a non-covalent complex, thereby producing a nanoparticle. 49. A method for making the nanoparticle of paragraph 34, comprising the steps of:

a) combining a conjugate of mPEG covalently bonded with a terminal amine of the PT- 91, wherein the ratio of mPEG molecules to PT-91 molecules is 1:1, with unconjugated PT-91 to form a mixture,- b) combining the mixture produced in step a) with siRNA such that the nucleic acid and the PT-91 form a non-covalent complex, thereby producing a nanoparticle; and

c) contacting the nanoparticle produced in step b) with Dimethyl 3 , 3 " -dithiobispropionimidate 2 HCl to produce a cross-linked nanoparticle.

50. A method for introducing a nucleic acid into a cell, comprising the step of contacting the cell with a nanoparticle of paragraph 1, paragraph 33 or

paragraph 35 or a composition of paragraph 44.

51. A method for reducing the expression of a target sequence in a cell, comprising the step of contacting the cell with a nanoparticle of paragraph 11 or paragraph 16.

52. A method for reducing the expression of a target sequence in a subject in need thereof,

comprising the step of administering a nanoparticle of paragraph 11 or paragraph 16 to the subject.

53. The nanoparticle of paragraph 1, wherein the molar ratio of conjugated cationic polymer:

unconjugated cationic polymer is a ratio of 1:9 to 9:1.

54. The nanoparticle of paragraph 53, wherein the molar ratio of conjugated cationic polymer:

unconjugated cationic polymer is a ratio of 1:1 to 3:1. 55. The nanoparticle of paragraph 1, wherein the nanoparticle has a mean diameter of less than 150 nm in 500 mM NaCl for at least 30 min. Brief Description of the Drawings

[0007] Figure 1 depicts the results of a reverse phase-HPLC (RP-HPLC) analysis performed with

unconjugated PT91 (Free PT) and purified mPEG-PT conjugate. Retention time in minutes is shown on the x-axis .

[0008] Figure 2 depicts the results of a MALDI-TOF analysis of the mPEG-PT conjugate. The darker line (6970) corresponds to the mPEG-PT conjugate. The lighter line (6969) corresponds to unconjugated PT91.

[0009] Figure 3 is a graph showing the effect of 100 mM NaCl on stability of NPX particles. Stability was determined by monitoring the effect of increased time in 100 mM NaCl solution on the effective diameter of NPX particles. The NPX particles tested were H3K4b, w/w 6; H3K4b, w/w 4; mPEG-H3K4b 5%, mPEG-H3K4b 10%;

mPEG-H3K4b 20%; and mPEG-H3K4b 46%. "w/w" is the weight : weight ratio of cationrnucleic acid. The percentages are molar percentages of PEG-PT to total PT (PEG-PT and free PT) .

[0010] Figure 4 is a graph showing the effect of crosslinking with DTBP on stability of NPX particles in 0.5 M NaCl. Stability was determined by monitoring the effect of increased time in 0.5 M NaCl solution on the effective diameter of NPX particles. The content of the mPEG-10kPT91 particles tested was 0; 20%; 20%, 10Ox DTBP; 50%; 65%; and 75%. The percentages are molar percentages of PEG-PT to total PT (PEG-PT and free PT) .

[0011] Figure 5 depicts the results of a

polyacrylamide gel analysis of NPX stability in serum. Stability was determined by monitoring siRNA

degradation by RNAse. Sample 1 - hVEGF siRNA in water, without RNAse inhibitors (Lane 1) . Sample 2 - hVEGF siRNA in serum with (Lane 2) and without (Lane 3) RNAse inhibitors. Sample 3 - un-crosslinked NPX of mPEG- PT/hVEGF siRNA in serum with (Lane 4) and without (Lane 5) RNAse inhibitors. Sample 4 - crosslinked NPX of mPEG-PT/hVEGF siRNA in serum with (Lane 6) and without (Lane 7) RNAse inhibitors.

[0012] Figure 6 depicts the results of a

polyacrylamide gel analysis of NPX stability in serum as measured by siRNA leakage. The varying DTBP/PT-91 molar ratios are listed across the top of the figure.

[0013] Figure 7A is a transmission electron

microscopy image showing the morphology of 65% mPEG- PT91/siRNA, w/w 4, IOOX DTBP NPX. Figure 7B is a transmission electron microscopy image showing the morphology of un-crosslinked PT91/hVEGF siRNA, w/w 4 NPX.

[0014] Figure 8 depicts NPX interaction with

polymorphonuclear leukocytes (PMNs) as measured by fluorescence-activated cell sorting (FACS) . The results obtained with naked crosslinked PT4-NPX (Figure 8A), NPX-mPEG(50%)XL (Figure 8B), NPX-mPEG (65%) XL

(Figure 8C) and NPX-mPEG (75%) XL (Figure 8D)) are shown.

[0015] Figure 9 depicts NPX interaction with

peripheral blood mononuclear cells (PBMCs) as measured by fluorescence-activated cell sorting (FACS) . The results obtained with naked crosslinked NPX (Figure 9A) and 65 PEGX (Figure 9B) are shown.

[0016] Figure 10 shows fluorescence microscopy images of EA.hy929 cells internalizing naked PT91 NPX (Figure 10A) , 50%mPEG-PT91 NPX (Figure 10B) , 65%mPEG- PT91 NPX (Figure 10C) and 75%mPEG-PT91 NPX (Figure 10D) . [0017] Figure 11 is a bar graph depicting the knockdown of hVEGF protein expression in PC-3 cells after treatment with various NPX containing hVEGF siRNA or mR2 (a negative control 25 bp blunt-ended double- stranded siRNA) .

[0018] Figure 12 is a bar graph depicting the knockdown of ApoB mRNA expression in BaI b/c mice systemically treated with PT-NPX containing ApoB siRNA. PT-NPX containing 25 bp blunt-ended double- stranded hVEGF siRNA served as a negative control.

[0019] Figure 13A and Figure 13B show the results of a polyacrylamide gel analysis of PEGX NPX before and after nebulization with Air Jet or I-NEB^® nebulizers. Intact hVEGF siRNA was used as a standard (lane 1) . Samples in lanes 3, 5 and 7 were treated with 0.1% SDS and 20 mM DTT to release siRNA from NPX completely.

[0020] Figure 14 depicts the serum stability of PEGX NPX containing various siRNAs. The following siRNAs were used: hV - hVEGF 25 bp blunt-ended siRNA,

unmodified; hV-MS - hVEGF 25 bp blunt-ended siRNA modified on every other nucleotide of both strands with 2 ' -0-methyl; Luc - Luciferase 25 bp blunt-ended siRNA, unmodified; Luc-OMe - Luciferase 25 bp blunt-ended siRNA, partially modified with 2 ' -0-methyl on all the uracils of one strand; ApoB - ApoB 25 bp blunt-ended siRNA, unmodified; ApoB-OMe - ApoB 25 bp blunt-ended siRNA, partially modified with 2 ' -0-methyl on all the uracils of one strand. Each NPX- siRNA sample was either treated or untreated with SDS and DTT.

[0021] Figure 15A and figure 15B depict the results of an RP-HPLC analysis of the stability of crosslinked 65% mPEG5k-PT91/hVEGF siRNA NPX. Figure 15A shows the percentage of free hVEGF siRNA (siRNA that was not encapsulated) as a function of time. Figure 15B shows the amount of total hVEGF siRNA (siRNA released after complete dissociation of the NPX) as a function of time.

[0022] Figure 16A is a line graph showing the siRNA concentration and siRNA recovery after concentration of mPEG5k-PT91/hVEGF siRNA w/w 4, 10Ox DTBP NPX by

centrifiltration. Figure 16B is a table showing the siRNA concentration, particle size and polydispersity after concentration of mPEG5k-PT91/hVEGF siRNA w/w 4, 10Ox DTBP NPX by centrifiltration . mPEG5k-PT91/hVEGF siRNA w/w 4, 10Ox DTBP NPX was concentrated using an Ultra-4 MWCO 5OkD (centrifuge at 3000 rpm, 15⁰C each time) . siRNA concentration was estimated using UV absorbance at 260nm. Particle size was measured following the protocol described in Example 2. Figures 16A and 16B show that the NPX can be concentrated by at least 30 fold with high siRNA recovery and unchanged particle size.

[0023] Figure 17 depicts the chemical structure of the PolyTran 91 (PT91) copolymer (NH₂-KHHH-KHHH-KHHHH- KHHHK)₄KKK-NH₂) .

[0024] Figure 18 depicts the chemical structure of the H3K4b copolymer (also referred to as PT-4) . Detailed Description of the Invention

[0025] The invention provides in one aspect a stable, non-toxic and biodegradable nucleic acid delivery vehicle that advantageously has a size that is suitable for in vivo use to deliver a nucleic acid to a target cell or tissue and that is able to maintain a suitable size under various conditions including in salt, serum, shear forces and high pressure such as during centrifugation. The delivery vehicle of the invention is in the form of a cross-linked nanoparticle comprising a nucleic acid-containing complex. The nanoparticle complex is also referred to herein as a "nanoplex" (NPX) . The NPX of the invention possesses additional advantages that will be described in detail herein and may be used for any application in which delivery of a nucleic acid to a cell or tissue is desired.

[0026] A nanoparticle or NPX of the invention comprises a nucleic acid, a cationic polymer, a

hydrophilic polymer and a cross-linking moiety.

[0027] The nucleic acid may be, for example, LNA, PNA, DNA, RNA or molecules comprising both

ribonucleotides and deoxyribonucleotides . The DNA may be any type of DNA including genomic DNA or cDNA. The RNA may be any type of RNA including but not limited to mRNA, tRNA, miRNA, tmRNA, rRNA, StRNA. The nucleic acid may be a large molecule such as an expression vector or may be an short oligonucleotide. Nucleic acids for use in a NPX of the invention include but are not limited to antisense nucleic acids including ribozymes, and aptamers . The nucleic acid may be single stranded or double stranded. In the case of double stranded nucleic acids, the strands may be completely complementary or may comprise one or more mismatches and, thus, be only partially complementary. In some embodiments, the nucleic acid is detectably labeled. Detectable labels include but are not limited to fluorescent labels and radiolabels, both of which will be will known to those of skill in the art.

[0028] The nucleic acids may comprise modified nucleotides including derivatives of purines and pyrimidines, non-natural nucleotides and chemically modified nucleotides. Chemically modified nucleotides may include but are not limited to nucleotides

comprising modifications such as 2 ' -0-methyl, 2'- O'alkyl, 2'-S-alkyl, 2'-fluoro-, 2' -halo, OMe (2'-O- methyl) , F (2 ' -fluoro) , ANA (altritol nucleic acid), HNA (hexitol nucleic acid), AEM (2 ' -aminoethoxymethyl) and APM (2 ' -aminopropoxymethyl) HM (4 ' -C-hydroxymethyl- DNA) , ADA (2' -N-adamantylmethylcarbonyl-20-amino-LNA (locked nucleic acid)) PYR (2 ' -N-pyren-l-ylmethyl-2 ' - amino-LNA) , EA (2 ' -aminoethyl) , GE (2 ' -guanidinoethyl) , CE (2' -cyanoethyl) , AP (2 ' -aminopropyl) , OX (oxetane- LNA) [, CLNA (2' ,4' -carbocyclic-LNA-locked nucleic acid), CENA (2 ' , 4 ' -carbocyclic-ENA-locked nucleic acid) ,AENA (2 ' -deoxy-2 ' -N, 4 ' -C-ethylene-LNA) .

[0029] Also within the invention are nanoplexes comprising nucleic acids with modified backbone

linkages. Numerous modified nucleic acid backbone linkages are known and a NPX of the invention may comprise any type of backbone linkage modification.

[0030] Such modified linkages may include, for example, phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates, phosphinates,

phosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates , thionoalkylphosphonates , thionoalkylphosphotriesters, morpholino linkages;

siloxane linkages; sulfide, sulfoxide and sulfone linkages; formacetyl and thioformacetyl linkages;

methylene formacetyl and thioformacetyl linkages;

alkene containing backbones; sulfamate containing backbones; methyleneimino and methylenehydrazino containing backbones; sulfonate and sulfonamide

containing backbones and amide containing backbones .

[0031] In embodiments comprising an anti-sense nucleic acid, the antisense nucleic acid is capable of hybridizing to a target nucleic acid such as but not limited to an mRNA, to reduce the expression or

activity of the target mRNA or a polypeptide encoded by the mRNA. In some embodiments, the NPX reduces the expression or activity of a target molecule by at least 10%. In some embodiments, the NPX reduces the

expression or activity of a target molecule by at least 20 %, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70% or at least 75%. In particular embodiments, the at least NPX reduces the expression or activity of a target molecule by at least 80%, at least 85%, at least 90%, at least 95% or to a level that is undetectable.

[0032] The antisense nucleic acid in some

embodiments is one that participates in RNA

interference (RNAi) such as short interfering RNA

(siRNA) or short hairpin RNA (shRNA) . In other

embodiments, the antisense nucleic acid participates in RNA activation (RNAa) such as short activating RNA (saRNA) . In siRNA embodiments, the nucleic acid may be single stranded or double stranded RNA (dsRNA) and may be from 15-50 nucleotides (nt) in length. In various embodiments, the nucleic acid is 15-30 nt, 15-25 nt, 16, 17, 18, 19, 20, 21, 22, 23 or 24 nt in length. The nucleic acid may comprise an 5' overhang of 1-4

nucleotides, a 3' overhang of 1-4 nucleotides, or both. In the case of a ds nucleotide, one or both strands may comprise such an overhang. In some embodiments the nucleotide is blunt-ended and in the case of ds nucleic acids, one or both strands may have a blunt end. In some embodiments, the nucleic acid is a ds, 25 base pair, blunt ended siRNA.

[0033] In some ds embodiments, the strength of the base-pairing in the 5' portion of the molecule is reduced compared to the strength of the base pairing in the 3 ' portion of the molecule as a result of one or more mismatches in the 5' portion. As used herein, the 5' portion of a double stranded nucleic acid is the half of the molecule starting from the 5' terminal nucleotide of the antisense strand. Accordingly, as used herein, the 3 ' portion of a double stranded molecule is the half of the molecule starting from the 3' terminal nucleotide of the antisense strand.

[0034] In some embodiments, the antisense nucleic acid comprises a nucleotide that creates a "wobble basepair" (e.g., G/U, I/U, I/A, and I/O when the antisense nucleic acid hybridizes with the target nucleic acid. See for example, United States patent 7,459,547, and US Patent Application Publications 2005/0186586, 2005/0181382 and 2006/0134787, which are incorporated herein by reference in their entirety for all purposes. In embodiments comprising a ds nucleic acid molecule, the ds molecule itself may comprise a wobble base pair.

[0035] A NPX of the invention may comprise two or more different nucleic acids. In some embodiments, the different nucleic acids may include a mixture of nucleic acids that do not contain chemical

modifications with nucleic acids that do contain chemical modifications. In some embodiments, the nucleic acids may have the same nucleotide sequence but may differ from each other by containing one or more chemical modifications. In the case of antisense nucleic acids, such as siRNA, the different antisense nucleic acids may target the same target sequence, may target different sequences in the same target nucleic acid and/or may target different nucleic acids.

[0036] In some embodiments, the nucleic acid is encodes a product that has a desired functional activity in a target cell or tissue upon transcription or translation in the cell. The desired product may be RNA or a polypeptide.

[0037] In a NPX of the invention, the cationic polymer forms a non-covalent complex with the

negatively charged nucleic acid. Suitable cationic polymers include but are not limited to

polyethyleneimine (PEI) , chitosan analogs, polylysine and polyornithine . According to the invention, the cationic polymer may be linear, branched or

dendrimeric .

[0038] In preferred embodiments, the cationic polymer is a co-polymer of histidine and a non- histidine amino acid that has a positive charge at a physiological pH . Such copolymers are known in the art, for example, in United States patents 7,163,695, 7,070,807 and 6,692,911, and in Leng, Q. et al . ,

"Histidine-Lysine Peptides as Carriers of Nucleic

Acids," Drug News Perspect 20(2), pp. 77-86 (2007), the contents of which are incorporated by reference herein in their entirety for all purposes. In some

embodiments, the cationic polymer is a histidine-lysine (HK) peptide copolymer. In various embodiments, the cationic polymer may be any one of: polymer selected from the group consisting of: a) CKHHH-KHHH-KHHH-KHHHKC (SEQ ID NO: 1);

b) (KHKHHKHHKHHKHHKHHKHK)₂K;

c) (CKHKHHKHHKHHKHHKHHKHK)₂K;

d) (KHHH-KHHH-KHHH-KHHHK)₄KKK;

e) KKK(KHHH-KHHH-KHHH-KHHHK) _4;

f) (CKHHH-KHHH-KHHH-KHHHK)₄KKK;

g) (KHHH-KHHH-KHHHH-KHHHK)₄KKK;

h) (KHHH-KHHHH-KHHHH-KHHHK)₄KKK;

i ) KKK ( KHHH-KHHHH-KHHHH-KHHHK) ₄ ;

j ) ( KHHH-KHHH-KHHH-KHHHK) BKKKKKKK ,

k) any one of a)-j) in which one or more lysines are replaced by an amino acid selected from the group consisting of: 2 , 5-diaminopentanoic acid, 2,4- diaminobutanoic acid and 2, 3-diaminopropionic acid; and

1) any one of a)-j) in which any of the core lysines is replaced by arginine, norarginine or ornithine. In particular embodiments, the HK copolymer is an H3K4b copolymer (Figure 18) or a PT91 copolymer (Figure 17) .

[0039] In various embodiments, the HK copolymer and the nucleic acid are present in the NPX at an HK copolymer to nucleic acid weight : weight ratio

(including HK copolymer counter ion) in the range of 1:1 to 20:1 and may be at an HK copolymer to nucleic acid weight : weight ratio of 2:1, 3:1, 4:1, 5:1, 6:1 or 8:1.

[0040] A NPX of the invention further comprises a hydrophilic polymer. Without being bound by theory, the hydrophilic polymer is believed to have a steric effect that may mask residual charge in the NPX and prevent aggregation of the NPX and non-specific

association of the NPX with molecules (such as those in serum) and with cells. The hydrophilic polymer may be any of a number of such polymers that decrease

aggregation of the formed nucleic acid nanoparticle and decrease non-specific binding, particularly in serum. Suitable hydrophilic polymers include but are not limited to polyethlyene glycol (PEG), poly (2'- methyloxyline) , poly (N-vinylpyrolidone) ,

[0041] In embodiments comprising polyethylene glycol (PEG) , the PEG may be of any suitable molecular weight and may be branched, unbranched or dendrimeric. In particular embodiments, the PEG has a molecular weight of from 2 KDa to 10 KDa, such as 3.4 KDa. 3.5 KDa, 5 KDa or 8 KDa. Monofunctional and bifunctional , including heterobifunctional PEG may be used in a NPX of the invention. By way of non- limiting example, mPEG- propionaldehyde (PAL) (such as mPEG-5 KDa-PAL or mPEG-10 KDa-PAL) , mPEG-vinyl sulfone (VS) (such as mPEG-5 KDa-VS) , NHS-PEG-VS, NHS-PEG-DEA, or any

alkylating or acylating PEG may be used in a NPX of the invention.

[0042] In some embodiments comprising polyethylene glycol (PEG) , the PEG is a heterobifunctional PEG reagent containing terminal protected (or "masked") aldehyde and ester functionalities. In certain

embodiments, the protected aldehyde functionality is an acetal or a thioacetal . In other embodiments, the protected ester functionality is an activated ester, including, but not limited to, an aromatic ester such as a para-nitrophenyl ester, or a heterocyclic ester such as an N-hydroxysuccinimide (NHS) ester.

Advantageously, the protected aldehyde and ester functionalities provide different reactivities such that a reaction, e.g., with a cationic polymer, can occur selectively at one terminus over the other.

[0043] In some embodiments comprising

heterobifunctional PEG reagents, the heterobifunctional PEG reagents provide two functional groups that can have different relative reactivities. In some

embodiments, one functional group may be more reactive than the other. In other embodiments, one functional group may prefer, or even be selective, for a

particular target molecule. In other embodiments, one functional group may selectively form a covalent bond with a target under certain reaction conditions.

[0044] In some embodiments, the PEG functional groups can be coupled to target molecules without activation. In other embodiments, the PEG functional groups are activated and then coupled to the target molecules.

[0045] In an NPX of the invention, the PEG is covalently linked (conjugated) to the cationic polymer. The covalent bonds may be stable covalent bonds, i.e., bonds that are stable under physiological conditions for at least 48 hours, such as amide, urethane, ether, secondary amine and thioether linkages, or labile covalent bonds, i.e., degradable under reducing

conditions. Non-limiting examples of labile linkages include disulfide, ester, acetal, orthoester, ketal and enzyme-cleavable bonds. In some embodiments, the PEG is conjugated to an amine of a copolymer of histidine and a non-histidine amino acid that has a positive charge at physiological pH . In some embodiments, the PEG is conjugated to a terminal amine of an HK

copolymer. In preferred embodiments, one mPEG molecule is conjugated to an HK copolymer molecule (a ratio of 1:1) .

[0046] In some embodiments, the NPX comprises cationic polymer that is conjugated to a hydrophilic polymer and also comprises unconjugated cationic polymer. According to the invention, the cationic polymer in the conjugate may be the same or different than the unconjugated cationic polymer. In particular embodiments, the NPX comprises unconjugated HK

copolymer and HK copolymer conjugated to PEG. The ratio of HK-PEG conjugate to unconjugated HK copolymer (molar percentage) in the NPX is at least 1:19, preferably a ratio of 1:9 to 9:1 and most preferably a ratio of 1:1 to 3:1. Illustrative ratios include and may be, for example 10:90. 30:70, 50:50, 65:35, 75:25, 85:15, 90:10. In preferred embodiments, the molar percentage of pegylated HK copolymer is from 50% to 90%.

[0047] An important feature of the NPX of the invention is the cross-linking. Any cross-linking agent may be used in the NPX of the invention as long as it is reactive with an amine group and is degradable in physiological conditions. Suitable cross-linking agents will be known to those of skill in the art.

Non-limiting examples of suitable cross-linking agents include Dimethyl 3 , 3 ' -dithiobispropionimidate• 2HCl (DTBP), Dithiobis (succinimidylpropionate) (DSP) , and 3 , 3 ' -dithiobis (sulfosuccinimidylpropionate) (DTSSP)

[0048] The extent of cross-linking that is desirable in a NPX of the invention will vary with the intended use, the components and the ratios of components of the NPX. Methods of achieving various amounts of cross- linking are known to those in the art and are useful along with methods described herein for producing an NPX. For example, different amounts of crosslinking may be achieved by reacting an uncrosslinked NPX with a cross-linking agent in different molar excess amounts compared to the cationic polymer. The crosslinking agent may be present during cross-linking in 20 to 200 molar excess, for example 50, 75 or 100 molar excess to the cationic polymer.

[0049] The NPX of the invention also may comprise additional components including a fusogenic moiety, a targeting moiety (including a nuclear targeting

moiety) , an endosomal lysing agent, an additional cationic polymer, an additional hydrophilic polymer, and/or additional cross-linking agents. In some embodiments, the same molecule may encompass one or more components. For example, the same molecule may be a fusogenic moiety and a cationic polymer or the same agent may be an endosomal lysing agent and a

hydrophilic polymer, and the like.

[0050] The NPX of the invention have a mean diameter of 40-150 nm, in various embodiments, the NPX have a mean diameter of about 50nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, about 80 nm, about 85 nm, about 90 nm about 95 nm, about 100 nm, about 110 nm, about 115 nm, about 120 nm, about 125 nm or about 130 nm. Those of skill in the art will know how to measure the size of the NPX, such as by light scattering techniques (including dynamic light scattering) or by microscopy, including electron microscopy, such as transmission electron microscopy. [0051] Advantageously, the mean diameter of the NPX of the invention does not vary substantially over time in the presence of NaCl and in serum. By "does not vary substantially" we mean that the mean diameter does not increase by more than 50%. In some embodiments, the mean diameter increases by less than 40%, less than 30%, less than 20%, less than 10% or does not increase at all.

[0052] Further advantages of the NPX include their ability to prevent or reduce the amount of RNAse- mediated degradation of the nucleic acid component of the NPX and the ability of the NPX to retain (or reduce leakage of) the nucleic acid in the NPX. The ability of the NPX of the invention to preserve the nucleic acid during storage or transport to a desired target site means that they advantageously deliver more active nucleic acid to the cell to enhance the desired effect, such as reducing the expression or activity of a target nucleic acid.

[0053] The invention further provides compositions, such as pharmaceutical compositions, comprising a NPX of the invention and a pharmaceutically acceptable carrier. In some embodiments, the compositions

comprise two or more different nanoplexes, for example, a first nanoplex comprising a nucleic acid that

hybridizes to a first target and a second nanoplex comprising a nucleic acid that hybridizes to a second target or comprising a nucleic acid that encodes a therapeutic product. In some embodiments, the

composition comprises one or more additional active ingredients, such as diagnostic or therapeutic agents.

[0054] The NPX of the invention and formulations thereof can be administered orally, topically, parenterally, by inhalation or spray or rectally in dosage unit formulations containing conventional nontoxic pharmaceutically acceptable carriers, adjuvants and vehicles. The term parenteral as used herein includes percutaneous, subcutaneous, intravascular (e.g., intravenous), intramuscular, intrathecal or local (including intratumoral) injection or infusion techniques and the like.

[0055] In a further aspect, the invention provides kits comprising a container, a composition comprising a NPX of the invention and instructions for use.

[0056] The invention further provides methods for producing and characterizing nanoplexes of the

invention that are described in detail in the Examples. According to the methods of the invention, in one step a hydrophilic polymer such as PEG is covalently linked to a cationic polymer, such as an HK copolymer to form, e.g., a PEG-HK copolymer conjugate. In a further step, unconjugated cationic polymer, such as unconjugated HK copolymer, is mixed with the conjugate formed in the previously described step in a ratio of at least 1:19 conjugated HK copolymer to unconjugated HK copolymer. The nucleic acid to be incorporated into the nanoplex is added to the conjugate-non-conjugate mixture under conditions that permit the non-covalent complexing of the nucleic acid with the cationic polymer such that the self-assembly of the nanoparticle is achieved.

Finally, the nanoparticle comprising the nucleic acid and the conjugated and unconjugated cationic polymer is cross-linked using an agent such as DTBP to produce a cross-linked nanoparticle (NPX) of the invention.

[0057] In a further aspect, the invention provides methods for using a NPX of the invention. The nanoplexes of the invention are useful in any

application in which delivery of a nucleic acid into a cell or to a tissue is desired including but not limited to gene therapy and delivery of agents that modulate the expression or activity of a target

molecule in a cell for diagnostic or therapeutic purposes in a subject in need thereof. In some

embodiments the subject is a human. In other

embodiments, the subject is a veterinary subject.

[0058] In some embodiments, the methods of the invention comprise use of a NPX of the invention to deliver a nucleic acid that reduces the expression and/or activity of a target nucleic acid in a cell in a subject in need thereof. In such an embodiment, the nucleic acid may be an antisense nucleic acid. In some embodiments, the method comprises a NPX in which the nucleic acid is siRNA for reducing the expression of a target mRNA via RNAi. In certain embodiments, the cell is a mammalian cell, in particular a human cell. In various embodiments, the cell is a tumor cell or an endothelial cell.

[0059] The nanoplexes of the invention may be used in methods for treating or preventing conditions and diseases including but not limited to autoimmune conditions and diseases, inflammatory conditions and diseases, infectious diseases, metabolic diseases, proliferative diseases including benign and malignant tumors, respiratory diseases, CNS conditions or

diseases and to treat or prevent undesirable

angiogenesis .

[0060] According to the methods of the invention, a NPX of the invention may be administered to a subject in combination with one or more additional active agents, including diagnostic and/or therapeutic agents. The additional active agent may be administered

simultaneously or sequentially with the NPX. In embodiments in which the NPX and additional agent are administered simultaneously, they may be coadministered or coformulated. Methods comprising the adminstration of a NPX of the invention may be part of a regimen that includes other treatment modalities including but not limited to surgery, radiation, nutritional therapy and photo therapy.

[0061] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be apparent to persons skilled in the art and are to be included within the and can be made without departing from the true scope of the invention .

Example 1

Preparation of mPEG-PT Conjugate

[0062] We dissolve histidine lysine copolymer such as PT4 (Figure 18) or PT91 (Figure 17) and polyethylene glycol such as mPEG-5KD-propionaldehyde (PEG- 5KD-PAL) , mPEG-lOKD- propionaldehyde (PEG-10KD-PAL) , or PEG-VS separately in phosphate buffer (0.1 ttiM, pH 5) at 40 mg/mL, then mix the two solutions at equal molar ratio to give a 2.75 mM solution of each component. To this solution we add NaCNBH₃ (5-fold excess) at 2-8⁰C. We allow the reaction to proceed at 2-8⁰C for 2 hours, then remove excess NaCNBH₃ in the reaction solution by dialysis against deionized water using a membrane with a molecular weight cut-off (MWCO) of 3000 Da.

Following the dialysis, we purify the remaining

reaction mixture with cationic exchange chromatography (IEX-HPLC) (column: HiPrep 16/10 CM FF column, mobile phase: A = 50 mM sodium phosphate (pH 7) ; B = 2M NaCl in 5OmM sodium phosphate (pH I)₁ flow rate: 2ml/min) . We combine and lyophilize the eluting fractions to give the desired mPEG-PT conjugate.

[0063] We analyze the purified mPEG-PT conjugate by reverse phase-HPLC (RP-HPLC) (column: Vydac RP C18,

250x4.6 mm, 5 urn, gradient: 0-50% mobile phase B for 35 minutes, mobile phase: A = 0.1% TFA in deionized water; B = 0.1% TFA in 95% acetonitrile and 5% deionized water, detection wavelength: UV 215nm, flow rate:

lml/min, room temperature) , MALDI-TOF (Applied

Biosystem, Voyager DE) , and NMR (JEOL ECX-400 NMR spectrometer, at 400MHz, in D₂O) .

[0064] RP-HPLC analysis of the purified conjugate mPEG5KD-PT91 (Figure 1) shows that this conjugate has a retention time of 38.5 min under the above HPLC

conditions. By comparison, the un-conjugated PT91 has a retention time of 27.9 min under the same conditions.

[0065] We also prepare mPEG-PT conjugates using PT4 and PT91 from different vendors with the same or different molar ratios of from 0.7 to 4.0 PEG: PT but preferably 1:1. We also prepare mPEG-PT conjugates using mPEGlOkD-PAL, mPEGlOkD-PAL and mPEG -VS.

[0066] MALDI-TOF analysis of the conjugate mPEG5KD- PT91 (Figure 2) shows that the conjugate has a

molecular weight of 14976 Da, indicating that, under the above conjugation conditions, there is one copy of mPEG-5KD conjugated to each PT91 molecule. Our NMR analysis shows that PT91:PEG-5KD ratio is about 0.8 to 1.2, which confirms this result.

Example 2

Preparation of PEGX Nanoparticles (PEGX NPX)

[0067] We receive siRNA, PT, and mPEG-PT as

lyophilized solids containing counter ions. We use weight ratio (including the counter ions) of PT to siRNA to define the nanoparticle compositions. We dissolve short interfering RNA (siRNA) in RNAse-free water at 0.4 mg/mL. We dissolve PT and mPEG-PT

separately in RNAse free water at desired

concentrations, and then we mix the PT and mPEG-PT solutions at a desired mPEG-PT:PT ratio (such as 5:95, 10:90, 30:70, 50:50, 65:35, 75:25, 85:15, 90:10) . To this mPEG-PT/PT mixture, we add equal volume of the siRNA solution (0.4 mg/mL) . We incubate the mixture at room temperature for 15 minutes, and then we adjust the pH of the mixture to 6.5 to form the mPEG-PT/PT

nanoplex (mPEG-PT/PT NPX) .

[0068] We crosslink the above NPX with crosslinking agent dimethyl 3 , 3 ' -dithiobispropionimidate• 2HCl

(DTBP) . We dissolve DTBP in sodium hydroxide solution (0.15N in water) at 30 mg/ml immediately prior to use, and then we mix the DTBP solution with the mPEG-PT/PT NPX suspension (as prepared above) at a desired molar ratio. Following that step, we adjust the pH of the mixture to 7.5. We allow the reaction to proceed at room temperature for 16 hours. We then adjust the pH of the reaction mixture to 6.5 to yield the desired crosslinked mPEG-PT/PT nanoplex (PEGX NPX) . Finally, we remove excess DTBP in the reaction solution by ultrafiltration or dialysis using a membrane with a MWCO of 100 kDa.

[0069] We characterize the NPX, crosslinked or un- crosslinked, by measuring its particle size, salt stability, serum stability, and particle morphology. These characterizations are discussed below.

(1) Particle size

[0070] We measure the particle size of NPX using Brookhaven ZeltaPALS Zeta Potential Analyzer

(Wavelength: 659.0 nm, Field Frequency: 2.00 Hz,

Voltage: 4.00 volts, Electric Field: 8.98 V/cm) .

[0071] Table 1 lists the particle sizes of several NPXs, which are prepared by varying the molar ratio of crosslinking agent to PT91. These NPXs all have a mPEG-PT:PT molar ratio of 65:35. As shown in Table 1, the crosslinked PEGX NPXs are generally smaller as compared to uncrosslinked NPXs. The smaller particle size is one of the advantages that the PEGX NPXs have. Table 1. Particle size of PEGX NPX crosslinked with DTBP

(2) Salt stability

[0072] We measure the stability of NPX particle in salt by monitoring their size changes in a salt solution. In a typical measurement, we add a NPX suspension (5 μL, 0.2 mg/ml siRNA) to a concentrated NaCl solution (50 μL) , and then we measure the particle size at 2 -minute intervals for 26 minutes.

[0073] As shown in Figure 3, NPX particles formed by naked PT and siRNA, and not cross-linked, such as

H3K4b, weight of PT/weight of siRNA (w/w) 4, and H3K4b, w/w 6, are not stable under the testing conditions. Their poor stability is characterized by the steady increase in the particle size in each case. Also shown in Figure 3, NPX particles that incorporated mPEG5KD- PT, such as mPEG-H3K4b 20% and mPEG-H3K4b 40%,

demonstrate much improved particle stability under the same testing conditions. The percentages here indicate the molar content of mPEG5KD-H3K4b in total H3K4b including mPEG5KD-H3K4b and free H3K4b.

[0074] As shown in Figure 4, the degree of

crosslinking also affects the stability of NPX

particles in salt. NPX particles with no or very low degree of crosslinking, such as mPEG10k-PT91 0% and mPEG10k-PT91 20%, demonstrate lessened stability, as compared with that of highly crosslinked NPX particles, such as mPEG10K-PT91 65% and mPEG10K-PT91 75%. The percentages here indicate the molar content of mPEGlOK- PT91 in total PT91 including mPEG10K-PT91 and free PT91.

(3) Serum stability

[0075] We evaluate the stability of NPX in serum by measuring the amount of siRNA degradation by RNAse, or by measuring the siRNA leakage.

[0076] In a typical assay of siRNA degradation by RNAse, we add NPX (0.5 μL, 0.2 gm/ml siRNA) to serum (9.5 μL) , in the presence or absence of an RNAse inhibitor (5 μL, SUPERase-In, Ambion, 20U/ul) . We incubate the mixture at 37⁰C for 1 hour, and then we analyze the mixture by polyacrylamide gel (PAGE) analysis using 20% TBE polyacrylamide gel at 160 V and 12 mA for 60 minutes.) . Degradation of siRNA is identified by migration of siRNA bands on PAGE.

[0077] As shown in Figure 5, crosslinked NPX of mPEG-PT/hVEGF siRNA is more stable against RNAse in serum than the un-crosslinked counterpart. In

particular, lane 5, which corresponds to an

uncrosslinked mPEG-PT/hVEGF siRNA NPX, shows that, in the absence of RNAse inhibitors, the majority of the hVEGF siRNA degrades in serum after 1 hour incubation. By contrast, under the same incubation conditions, a large fraction of hVEGF siRNA remains intact as shown in lane 7, which corresponds to a crosslinked mPEG- PT/hVEGF siRNA NPX. This result indicates that

crosslinked NPX can protect siRNA from RNAse

degradation in serum.

[0078] In a typical assay of siRNA leakage in serum, we add NPX (0.5 μL, 0.2mg/ml siRNA) to serum (9.5 μL) and an RNAse inhibitor solution (5 μL, SUPERase-In,

Ambion, 20U/ul) . We incubate the mixture at 37⁰C for 1 hour, and then we split the mixture equally into two microcentrifuge tubes. To the first tube, we add RNase free water (4 μL) . To the second tube, we add DTT (200 mM, 2 μL) , and then we incubate the mixture for 15 minutes at room temperature. Following that step, we add SDS (1%, 2 μL) to the second tube, and then we incubate the resulting mixture for another 15 minutes. Finally, we analyze samples from both tubes by

polyacrylamide gel (PAGE) analysis using 20% TBE polyacrylamide gel at 160 V and 12 mA for 60 minutes. The lane resulted from the first tube (without SDS or DTT) is called "as is" and the lane resulted from the second tube (with SDS and DTT) is called "total

release" .

[0079] As shown in Figure 6, siRNA leakage from crosslinked 65%mPEG-PT/siRNA, W/W 4, 10Ox DTBP

nanoparticle (65%mPEGX NPX) decreases with increase of DTBP/PT molar ratio. The siRNA leakage is estimated by the ratio of intensity of the band without SDS or DTT: intensity of the band with SDS and DTT. The intensity of each band is measured by densitometry (Table 2) . This result also confirms that the degree of

crosslinking is increased with increase of DTBP.

Table 2. Densitometry readings of bands depicted in Figure 6.

[0080] The uncrosslinked NPX disintegrated in serum. This is probably due to the interactions between the NPX and serum proteins and electrolytes . The

disintegration results in releasing of the siRNA, which is then degraded by RNAse in the absence of the RNAse inhibitors. In contrast, crosslinked NPX is resistant to the disintegration and therefore is able to retain the siRNA within the NPX, preventing degradation from the RNAse .

(4) Transmission electron microscopy (TEM)

[0081] We characterize the morphology (and the size) of NPX particles using TEM. In a typical TEM study, we coat the copper grid (400-mesh) with a layer of ultrathin carbon, and then we clean the grid by plasma glow discharge. We add a NPX suspension (6 μL,

0.2mg/mL siRNA) on the grid, and then we add PTA (6 μL, pH 7.0) to stain the NPX particles. After one minute, we blot the grid using a filter paper, and then we take the TEM images .

[0082] As shown in Figure 7, crosslinked 65%mPEGX NPX preserves the spherical morphology of uncrosslinked naked PT91/siRNA NPX. In both cases, we use the same siRNA and PT/siRNA ratio. The average particle size of crosslinked 65PEGX NPX is about 60 nm, which is in good agreement with that measured by dynamic light

scattering (DLS result for 65PEGX NPX : particle size 70.3 nm, polydispersity 0.019).

(5) Stability of crosslinked NPX

[0083] The stability of NPX may be measured over time by RP-HPLC. For example, we measured the

stability of crosslinked 65% mPEG5k-PT91/hVEGF siRNA NPX. The NPX was stored at 4-8°C. Its properties were analyzed periodically. The particle size was analyzed using dynamic light scattering with a protocol

described above. Free siRNA (siRNA that was

unencapsulated) (Figure 15A) and total siRNA (released from NPX after complete dissociating the NPX) (Figure 15B) were analyzed using RP-HPLC. The HPLC conditions were as follows: column: Luna-amino, 5m, 4.6x150 mm at 25 ⁰C, flow: 1 mL/min, UV/Vis detection, gradient elution from 20 to 60% B in 30 min where mobile phase A (MPA) is 1OmM TRIS-HCl, 10% MeOH at pH 7 and mobile phase B (MPB) is 2M NaCl in MPA. As shown in Table 3, the particle size remained stable over time. Table 3 . Particle si ze of crossl inked 65 % mPEG5k- PT91/hVEGF siRNA NPX over time

Example 3

NPX interaction with PMN and PBMC

[0084] We anti-coagulate a fresh human blood sample using ACD (citric acid/sodium citrate/dextrose) , and then we separate red blood cells (RBCs) and white blood cells (WTCs) from the blood sample by dextran

sedimentation. Following that step, we remove RBCs by hypotonic lysis, and then we use Ficol sedimentation to isolate Polymorphonuclear leukocytes (PMNs) and

peripheral blood mononuclear cells (PBMCs) . We then incubate NPX containing Alexa488-labeled siRNA with

PMNs or PBMCs for 1 hour. Following the incubation, we treat the cells with heparin and tris(2- carboxyethyDphosphine (TCEP), and then we wash the cells to remove external NPX particles. Finally, we use Fluorescence-activated cell sorting (FACS) analysis to evaluate NPX internalization.

[0085] As shown in Figures 8 and 9, incorporation of mPEG-PT into the crosslinked NPX effectively suppresses the interactions of the NPX with PMNs and PBMCs. By contrast, crosslinked naked PT/siRNA NPX, which is positively charged, interacts strongly with PMNs and PBMCs and, therefore, can be internalized readily, possibly leading to poor target specificity and fast clearance.

Example 4

NPX Internalization

[0086] We seed EA.hy926 cells onto a 24-well plate (Costa, Corning Incorporated, NY) a day before the internalization experiment. We add NPXs containing Alexa488-labeled siRNA to the cells at 10 μg/mL, and then we incubate the cells for 3 hours in the presence of 10% fetal bovine serum (FBS) . Following the

incubation, we replace the cell culture medium with fresh DEME supplemented with 10% FBS to remove external NPX, and then we take fluorescence images of the cultured cells under microscope.

[0087] The fluorescence images shown in Figure 10 indicate that EA.hy926 cells can effectively

internalize crosslinked naked PT91 NPX. Figure 10 also shows that EA.hy926 cells can internalize crosslinked PEGX NPX to a similar extent, as compared to that of crosslinked naked PT91 NPX. The percentages here indicate the molar content of mPEG-PT91 in total PT91 including mPEG-PT91 and free PT91. Further, PEGX NPX demonstrates a more diffused distribution pattern within the cells, as compared to the punctuated

distribution pattern of the naked PT91 NPX. This finding indicates that PEGX NPX is able to internalize, and also escape from endosomes, which is a critical step for siRNA to take effect. In contrast, naked PT91 NPX appears in endosomes after being taken up. Example 5

Knockdown of hVEGF by NPX in PC-3 Cells

[0088] We trypsinize PC-3 cells, and then seed the cells onto a 48-well plate (Costa, Corning

Incorporated, NY) a day prior to use. We add NPX into each well, and then incubate the plate for 48 hours. Following the incubation, we replace the cell culture medium with fresh complete medium (RPMI 1640,

supplemented with 10% FBS) , and then we collect the cell supernatant at 72 hr for hVEGF measurement by

ELISA assay (R&D Cat# DY293B) (ELISA data were shown in Fig. H)] We wash cells gently with cold PBS buffer (Mediatech Inc., Cat# 21-031), then we lyse cells using RIPA buffer. Next, we transfer the lysate from each well into an Eppendorf tube, and then we centrifuge the tubes to remove cell pellets. We take the supernatant from each tube for total protein measurement using a bicinchoninic acid (BCA) protein assay kit (Pierce, Cat# 23225) .

[0089] The hVEGF protein measurement as shown in Figure 11 indicates that crosslinked PEGX NPX

containing hVEGF siRNA can effectively knockdown hVEGF expression by over 90%.

Example 6

Jn vivo Knockdown of ApoB

[0090] We administer 3 mg/kg of crosslinked mPEG5k- PT91/ApoB-siRNA, w/w 4, 10Ox DTBP NPX to 5 BaI b/c mice (Treatment Group) by intravenous injection on day 1 and day 2. On day 4, we harvest 2 liver samples from each animal in the Treatment Group and from each of 3 BaI b/c mice in the Control Group (no treatment) . We isolate the total RNA from each liver sample, and then we measure the levels of Apo mRNA using quantitative real-time polymerase chain reaction (qRT-PCR) assay. We calculate the relative levels of ApoB mRNA using the average of untreated control group as 100%.

[0091] As shown in Figure 12, mice in the Treatment Group demonstrated a significant knockdown of ApoB in the liver. Figure 12 also demonstrates a dose- dependent knockdown of ApoB in vivo. Comparing to similar NPX containing hVEFG-siRNA as a control, the (PEGX) NPX containing ApoB siRNA is able to knockdown more than 40% of ApoB expression.

Example 7

Nebulization of PEGX NPX

[0092] We prepare crosslinked PEGX NPX containing hVEGF siRNA according to the protocols described in Examples 1 and 2, and then we nebulize the NPX using Air Jet or I-NEB^® nebulizer. We then collect and analyze the nebulized NPX.

[0093] Our analysis shows that there is little change in particle size of 65%mPEGX NPX after

nebulization by either nebulizer (Table 4) . The majority of siRNA is still encapsulated in the 65%mPEGX NPX after nebulization (Figure 13); therefore,

nebulization does not affect the integrity of PEGX NPX. Table 4. Particle size of PEGX NPX before and after nebulization

Example 8

NPX Made with Various siRNAs

[0094] Following the protocols described in Examples 1 and 2, we prepare crosslinked PEGX NPXs with various siRNAs, and measure their size in HEPES buffers and retention of siRNAs in serum.

[0095] All of the 65%mPEGX NPXs have very similar particle size, and all of them are smaller than 120 nm (Table 5) . Our study also shows that these 65%mPEGX NPXs have very low siRNA leakage and excellent serum stability (Figure 14) . Figure 14 presents data

generated with 65%mPEGX NPX containing various siRNAs. All the siRNAs used in this study contain 25 bps . In Figure 14, the first lane of each sample 1-6 represents an untreated 65%mPEGX NPX; the second lane of each sample represents a 65%mPEGX NPX treated with DTT and SDS. Figure 14 demonstrates that all of the untreated 65%mPEGX NPXs retain the siRNA in serum, regardless of the sequence of the siRNA or the presence of any modification.

Table 5. Particle sizes of PEGX NPX containing various siRNA

[0096] All publications, patents, and patent applications cited in this specification are incorporated herein by reference.

Claims

Claims We claim:

a) a mean diameter of less than 150 nm;

b) a mean diameter of less than 150 nm in 100 mM NaCl for at least 30 min;

c) a mean diameter of less than 150 nm after 30- fold concentration;

d) retains the nucleic acid in serum; and

e) internalizes in a target cell.

2. The nanoparticle of claim 1, wherein the nucleic acid is selected from the group consisting of: DNA, LNA, RNA, DNA-RNA hybrids, PNA.

3. The nanoparticle of claim 1, wherein the nucleic acid is RNA and is selected from the group consisting of: mRNA, miRNA, tRNA, tmRNA, rRNA and StRNA.

4. The nanoparticle of claim 1, wherein the nucleic acid is an antisense nucleic acid.

5. The nanoparticle of claim 4, wherein the antisense nucleic acid is selected from the group consisting of: siRNA, shRNA, miRNA, ribozymes .

6. The nanoparticle of claim 1, wherein the nucleic acid comprises a modified backbone linkage selected from the group consisting of: phosphorothioate linkages, phosphoramidate linkages, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates, phosphinates, phosphoramidates and aminoalkylphosphoramidates , thionophosphoramidates , thionoalkylphosphonates , thionoalkylphosphotriesters , morpholino linkages; siloxane linkages; sulfide, sulfoxide and sulfone linkages; formacetyl and

linkages; sulfonate and sulfonamide linkages and amide linkages.

7. The nanoparticle of claim 1, wherein the nucleic acid comprises a modified nucleotide selected from the group consisting of: a purine derivative, a pyrimidine derivative, non-natural nucleotides, nucleotides comprising modifications such as 2'-O- methyl, 2'-0' alkyl, 2'-S-alkyl, 2'-fluoro-, 2' -halo, OMe (2' -0-methyl) , F (2'-fluoro), ANA (altritol nucleic acid), HNA (hexitol nucleic acid), AEM (2'- aminoethoxymethyl) and APM (2 ' -aminopropoxymethyl) HM (4' -C-hydroxymethyl-DNA) , ADA (2'-N- adamantylmethylcarbonyl-20-amino-LNA (locked nucleic acid)) PYR (2 ' -N-pyren-l-ylmethyl-2 ' -amino-LNA) , EA (2' -aminoethyl) , GE (2 ' -guanidinoethyl) , CE (2'- cyanoethyl) , AP (2 ' -aminopropyl) , OX (oxetane-LNA) [, CLNA (2' ,4' -carbocyclic-LNA-locked nucleic acid), CENA (2 ' , 4 ' -carbocyclic-ENA-locked nucleic acid) ,AENA (2'- deoxy-2' -N, 4' -C-ethylene-LNA) .

8. The nanoparticle of claim 1, wherein the nucleic acid is single stranded.

9. The nanoparticle of claim 1, wherein the nucleic acid is double stranded.

10. The nanoparticle of claim 1, wherein the nucleic acid comprises:

a) an overhang of 1-4 nucleotides;

b) two strands each with an overhang of 1-4 nucleotides ;

c) a blunt end; or

d) two strands with blunt ends.

11. The nanoparticle of claim 1, wherein the nucleic acid is an antisense nucleic acid having a length of 15-30 nucleotides.

12. The nanoparticle of claim 1, wherein the nucleic acid is an antisense nucleic acid having a length of 19-30 nucleotides.

13. The nanoparticle of claim 1, wherein the nucleic acid is an antisense nucleic acid having a length of 25 nucleotides.

14. The nanoparticle of claim 11, wherein the nucleic acid is an antisense nucleic acid and wherein the nanoparticle internalizes in a cell and reduces expression of a target molecule by at least 10%.

15. The nanoparticle of claim 14, wherein the expression of a target molecule is reduced by at least 50 %.

16. The nanoparticle of claim 1, wherein the nucleic acid is siRNA.

17. The nanoparticle of claim 1, wherein the nucleic acid is 25 base pair, blunt-ended double stranded siRNA.

18. The nanoparticle of claim 1, wherein the hydrophilic polymer is selected from the group

19. The nanoparticle of claim 18, wherein the hydrophilic polymer is PEG and wherein the PEG is selected from the group consisting of linear PEG, branched PEG and dendrimeric PEG.

20. The nanoparticle of claim 19, wherein the PEG is selected from the group consisting of:

monofunctional PEG, homobifunctional PEG and

heterobifunctional PEG.

21. The nanoparticle of claim 20, wherein the PEG is a heterobifunctional PEG comprising a terminal acetal or thioacetal .

22. The nanoparticle of claim 20, wherein the PEG is a heterobifunctional PEG comprising a terminal ester .

23. The nanoparticle of claim 19, wherein the PEG is an alkylating or acylating PEG.

24. The nanoparticle of claim 19, wherein the PEG has a formula weight selected from the group consisting Of: 2 KD, 3.5 KD, 5 KD 8 KD and 10 KD.

25. The nanoparticle of claim 1, wherein the hydrophilic polymer is covalently joined to the

cationic polymer via bond selected from the group consisting of: amide, urethane, ether, secondary amine, thioether, disulfide, ester, acetal, orthoester, ketal and enzyme-cleavable bonds.

26. The nanoparticle of claim 1, wherein the covalent bond is stable under physiological conditions.

27. The nanoparticle of claim 1, wherein the cationic polymer is selected from the group consisting of: polyethyleneimine (PEI), a co-polymer of histidine and a non-histidine amino acid that has a positive charge at a physiological pH, chitosan analogs,

polylysine, polyornithine .

28. The nanoparticle of claim 26, wherein the cationic polymer is a co-polymer of histidine and a non-histidine amino acid that has a positive charge at a physiological pH and is selected from the group consisting of: a linear co-polymer, a branched copolymer, a dendrimeric co-polymer.

29. The nanoparticle of claim 1, wherein the cationic polymer is a polymer selected from the group consisting of:

a) CKHHH-KHHH-KHHH-KHHHKC (SEQ ID NO: 1 ) ;

b) (KHKHHKHHKHHKHHKHHKHK)₂K;

C) (CKHKHHKHHKHHKHHKHHKHK)₂K;

d) (KHHH-KHHH-KHHH-KHHHK)₄KKK;

e) KKK(KHHH-KHHH-KHHH-KHHHK) ₄;

f) (CKHHH-KHHH-KHHH-KHHHK)₄KKK;

g) (KHHH-KHHH-KHHHH-KHHHK)₄KKK;

h) (KHHH-KHHHH-KHHHH-KHHHK)₄KKK;

i) KKK (KHHH-KHHHH-KHHHH-KHHHK) ₄;

j ) (KHHH-KHHH-KHHH-KHHHK) _SKKKKKKK;

1) any one of a)-j) in which any of the core lysines is replaced by arginine, norarginine or ornithine .

30. The nanoparticle of claim 1, wherein the cationic polymer in said hydrophilic polymer-cationic polymer conjugate and said unconjugated cationic polymer are different cationic polymers.

31. The nanoparticle of claim 1, wherein said composition is formed by combining said hydrophilic polymer-cationic polymer conjugate with said

unconjugated cationic polymer in a molar ratio of at least 1:19.

32. The nanoparticle of claim 1, wherein said cross-linking agent is selected from the group

consisting of: Dimethyl 3 , 3 " -dithiobispropionimidate (DTBP), Dithiobis (succinimidylpropionate) (DSP) and 3, 3' -dithiobis (sulfosuccinimidylpropionate) (DTSSP) .

33. The nanoparticle of claim 1, wherein the mean diameter is 100 nm or less.

34. The nanoparticle of claim 16, comprising two or more two different siRNAs, wherein the different siRNAs are selected from the group consisting of:

a) siRNAs that target a different sequence in the same target nucleic acid;

b) siRNA's that target different target nucleic acids,- and

c) siRNAs with and without a chemical

modification.

35. The nanoparticle of claim 1, wherein the nucleic acid is siRNA, the hydrophilic polymer is mPEG, the cationic polymer is HK copolymer PT- 91, wherein said composition comprises: a) a conjugate of mPEG covalently bonded with an amine of HK copolymer PT- 91, wherein the ratio of mPEG molecules to HK copolymer PT- 91 molecules is 1:1; and

b) unconjugated HK copolymer PT-91.

36. The nanoparticle of claim 35, wherein said composition further comprises cross-linking.

37. The nanoparticle of claim 35, wherein said composition is formed by combining the mPEG-PT-91 conjugate with unconjugated PT-91 in a molar ratio of at least 1:19.

38. The nanoparticle of claim 37, wherein the mPEG is selected from the group consisting of: mPEG 2K, mPEG 3.4K, mPEG 5K, mPEG 8K and mPEG 1OK.

39. The nanoparticle of claim 35, produced by a method comprising the steps of

unconjugated PT-91 to form a mixture of conjugated and unconjugated PT-91;

40. The nanoparticle of claim 35, wherein in step a) , the conjugated and unconjugated PT-91 are combined at a molar ratio of at least 1:19.

41. The nanoparticle of claim 35, further

comprising the step:

42. The nanoparticle of claim 39, wherein the nanoparticle produced in step b) is contacted with 50, 75 or 100 molar excess of dithiobispropionimidate 2 HCl to PT-91.

43. The nanoparticle of claim 1 or claim 35, wherein the nucleic acid is detectably labeled.

44. The nanoparticle of claim 37, wherein the siRNA is detectably labeled.

45. The nanoparticle of claim 1, claim 34 or claim 37, further comprising one or more components selected from the group consisting of: a fusogenic molecule, a targeting moiety and an endosomal lysing agent .

46. A composition comprising a nanoparticle of claim 1, claim 35 or claim 37 and a pharmaceutically acceptable carrier.

47. The composition of claim 44, further

comprising an additional active component which is a therapeutic agent.

48. A kit comprising a container, the

nanoparticle of claim 1, claim 35 or claim 37 or the composition of claim 46 and instructions for use.

49. A method for making the nanoparticle of claim 1, comprising the steps of:

c) contacting the nanoparticle produced in step b) with a cross-linking agent to produce a cross-linked nanoparticle.

50. A method for making the nanoparticle of claim

35, comprising the steps of:

b) combining the mixture produced in step a) with siRNA such that the nucleic acid and the PT- 91 form a non-covalent complex, thereby producing a nanoparticle.

51. A method for making the nanoparticle of claim

36, comprising the steps of:

a) combining a conjugate of mPEG covalently bonded with a terminal amine of the PT- 91, wherein the ratio of mPEG molecules to PT-91 molecules is 1:1, with unconjugated PT-91 to form a mixture,- b) combining the mixture produced in step a) with siRNA such that the nucleic acid and the PT- 91 form a non-covalent complex, thereby producing a nanoparticle; and

52. A method for introducing a nucleic acid into a cell, comprising the step of contacting the cell with a nanoparticle of claim 1, claim 35 or claim 37 or a composition of claim 46.

53. A method for reducing the expression of a target sequence in a cell, comprising the step of contacting the cell with a nanoparticle of claim 11 or claim 16.

54. A method for reducing the expression of a target sequence in a subject in need thereof,

comprising the step of administering a nanoparticle of claim 11 or claim 16 to the subject.

55. The nanoparticle of claim 1, wherein the molar ratio of conjugated cationic polymer:

unconjugated cationic polymer is a ratio of 1:9 to 9:1.

56. The nanoparticle of claim 55, wherein the molar ratio of conjugated cationic polymer:

unconjugated cationic polymer is a ratio of 1:1 to 3:1.

57. The nanoparticle of claim 1, wherein the nanoparticle has a mean diameter of less than 150 nm in 500 mM NaCl for at least 30 min.