WO2015179492A1 - Shape-controlled nucleic acid nanoparticles for in vivo delivery of nucleic acid therapeutics - Google Patents

Abstract

Methods for preparing nucleic acid nanoparticles having a controllable shape and their use for delivering nucleic acids to a subject in need of treatment of a neurological disease are disclosed.

Description

SHAPE-CONTROLLED NUCLEIC ACID NANOPARTICLES FOR IN VIVO DELIVERY OF NUCLEIC ACID THERAPEUTICS

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No.

62/000,838, filed May 20, 2014, which is incorporated herein by reference in its entirety.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under R21EB015152 awarded by the National Institutes of Health (NIH), W81XWH-10-2-0053 awarded by the U.S. Army Defense Threat Reduction Agency (DTRA), and R01NS041438 awarded by the National Institute of Neurological Disorders and Stroke (NINDS). The government has certain rights in the invention. iNCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED

ELECTRONICALLY

This application contains a sequence listing. It has been submitted electronically via EFS-Web as an ASCII text file entitled "111232-00384_ST25.txt". The sequence listing is 1,490 bytes in size, and was created on May 18, 2015. It is hereby incorporated by reference in its entirety.

BACKGROUND

Identifying parameters that mediate efficient delivery of siRNA via nanoparticles to the central nervous system (CNS) can have important therapeutic implications for treating CNS-related diseases, such as Alzheimer's Disease, a progressive degenerative brain disorder characterized pathologically by the accumulation of amyloid-B (AB) species and neurofibrillary tangles. B-site APP cleaving enzyme 1 (BACE1), a key enzyme required for the generation of AB from the amyloid-B precursor protein (APP), is a well-validated therapeutic target amenable to the RNA interference therapeutic strategy.

Considerable progress has been made toward developing RNA interference as a therapeutic strategy (Alvarez-Erviti et al, 2011). Efficient delivery and knockdown of suitable targets in an in vivo context has been challenging, however. The development of siR A loaded nanoparticles has demonstrated their capability for packaging and delivery in in vitro and in vivo models (Lee et al, 2012; Jensen et al, 2013). Various polycationic carriers have the capability to package and deliver siRNA in the form of nanoparticles, although their in vivo transfection efficiency has been disappointing.

Studies have shown that the shape of the nanoparticles can play a key role toward improving delivery efficiency (Jiang et al, 2013). The effort to build a viable in vivo siRNA delivery method will involve the need to understand the effect of varying shape on payload delivery, while maintaining the integrity of the nanoparticle complex until it reaches its site of delivery. Thus far, controlling the shape of these siRNA nanoparticles, however, has been inaccessible.

SUMMARY

The practice of the present invention will typically employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant nucleic acid (e.g., DNA) technology, immunology, and RNA interference (RNAi) which are within the skill of the art. Non- limiting descriptions of certain of these techniques are found in the following publications: Ausubel, F., et al, (eds.), Current Protocols in Molecular Biology, Current Protocols in Immunology, Current Protocols in Protein Science, and Current Protocols in Cell Biology, all John Wiley & Sons, N.Y., edition as of December 2008; Sambrook, Russell, and Sambrook, Molecular Cloning. A Laboratory Manual, 3^rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2001; Harlow, E. and Lane, D., Antibodies— A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1988; Freshney, R. I., "Culture of Animal Cells, A Manual of Basic Technique", 5th ed., John Wiley & Sons, Hoboken, N.J., 2005. Non-limiting information regarding therapeutic agents and human diseases is found in Goodman and Gilman's The Pharmacological Basis of Therapeutics, 11th Ed., McGraw Hill, 2005, Katzung, B. (ed.) Basic and Clinical Pharmacology, McGraw-Hill/Appleton & Lange 10^th ed. (2006) or 11th edition (July 2009). Non-limiting information regarding genes and genetic disorders is found in McKusick, V. A.: Mendelian Inheritance in Man. A Catalog of Human Genes and Genetic Disorders. Baltimore: Johns Hopkins University Press, 1998 (12th edition) or the more recent online database: Online Mendelian Inheritance in Man, OMIM™. McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, Md.) and National Center for Biotechnology Information, National Library of Medicine (Bethesda, Md.), as of May 1, 2010, World Wide Web URL: http://www.ncbi.nlm.nih.gov/omim/ and in Online Mendelian Inheritance in Animals (OMIA), a database of genes, inherited disorders and traits in animal species (other than human and mouse), at

http://omia.angis.org.au/contact.shtml. All patents, patent applications, and other publications (e.g., scientific articles, books, websites, and databases) mentioned herein are incorporated by reference in their entirety. In case of a conflict between the specification and any of the incorporated references, the specification (including any amendments thereof, which may be based on an incorporated reference), shall control. Standard art-accepted meanings of terms are used herein unless indicated otherwise. Standard abbreviations for various terms are used herein.

In some aspects, the presently disclosed subject matter provides a method for preparing a shape-controlled nucleic acid nanoparticle, the method comprising:

providing a copolymer solution comprising a block copolymer or a graft copolymer of a water soluble, non-charged polymer and a polycation in a first solvent, wherein the water soluble, non-charged polymer and the polycation each have a structure and a molecular weight; and mixing the copolymer solution with a solution of nucleic acid at a predetermined nucleic acid to copolymer ratio and pH in a second solvent to form a shape-controlled nucleic acid nanoparticle, wherein the nucleic acid has a predetermined number of base pairs and the first and second solvent can be the same or different; and wherein the shape-controlled nucleic acid nanoparticle has a shape controlled by one or more of the: structure of the water soluble, non-charged polymer; molecular weight of the water soluble, non-charged polymer; structure of the polycation; molecular weight of the polycation; number of base pairs of the nucleic acid; ratio of nucleic acid to copolymer; pH; first solvent comprising the copolymer solution and/or the second solvent comprising the nucleic acid solution, and, if the copolymer is a graft copolymer, a graft density thereof.

In other aspects, the presently disclosed subject matter provides a method for preparing a shape-controlled siRNA nanoparticle, the method comprising: providing a copolymer solution comprising a block copolymer or a graft copolymer of a water soluble, non-charged polymer and a polycation in a first solvent, wherein the water soluble, non-charged polymer and the polycation each have a structure and a molecular weight; and mixing the copolymer solution with a solution of siRNA at a predetermined siRNA to copolymer ratio and pH in a second solvent to form a shape- controlled siRNA nanoparticle, wherein the siRNA has a predetermined number of base pairs and the first and second solvent can be the same or different; and wherein the shape-controlled siRNA nanoparticle has a shape controlled by one or more of the: structure of the water soluble, non-charged polymer; molecular weight of the water soluble, non-charged polymer; structure of the polycation; molecular weight of the polycation; number of base pairs of the siRNA; ratio of siRNA to copolymer; pH; first solvent comprising the copolymer solution and/or the second solvent comprising the siRNA solution, and, if the copolymer is a graft copolymer, a graft density thereof.

In particular aspects, the water soluble, non-charged polymer comprises polyethylene glycol (PEG) and the polycation is linear polyethylenimine (LPEI).

In certain aspects, the presently disclosed subject matter provides a method for treating a disease or condition, the method comprising administering to a subject in need of treatment thereof, a shape-controlled nucleic acid nanoparticle described hereinabove, or a pharmaceutical composition thereof, in an amount effective for treating the disease or condition.In other aspects, the presently disclosed subject matter provides a method for treating a disease or condition, the method comprising administering to a subject in need of treatment thereof, a shape-controlled siRNA nanoparticle described hereinabove, or a pharmaceutical composition thereof, in an amount effective for treating the disease or condition.

In particular aspects, the disease is a neurological disease. In yet more particular aspects, the neurological disease is selected from the group consisting of Amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Parkinson's disease, Alzheimer's disease, Huntington's disease, and dementia with Lewy Bodies.

Certain aspects of the presently disclosed subject matter having been stated hereinabove, which are addressed in whole or in part by the presently disclosed subject matter, other aspects will become evident as the description proceeds when taken in connection with the accompanying Examples and Figures as best described herein below. BRIEF DESCRIPTION OF THE FIGURES

Having thus described the presently disclosed subject matter in general terms, reference will now be made to the accompanying Figures, which are not necessarily drawn to scale, and wherein:

FIG. 1A and FIG. IB show LPEI_17k-2% PEGio_k/siRNA nanoparticles, N/P 5:

FIG. 1A shows short rods, made in 70% DMF. Scale bar 500 nm; and FIG. IB shows worms, after two weeks. Scale bar = 2 μιη;

FIG. 2A and FIG. 2B show LPEI_17k -4% PEGi_0k /siRNA nanoparticles, N/P 5: FIG. 2A shows worms, made with siRNA targeting amyloid-B precursor protein (sequence A). Scale bar = 2 μιη; and FIG. 2B shows short rods, made with siRNA targeting B-site APP cleaving enzyme 1 (sequence B). Scale bar = 500 nm;

FIG. 3 A, FIG. 3B, FIG. 3C and FIG. 3D show LPEI_17k-2% PEGio_k/siRNA nanoparticles: FIG. 3 A shows N/P 4, worms. Scale bar = 1 μιη; FIG. 3B shows N/P 5, short rods and a few long worms. Scale bar = 1 μιη; FIG. 3C shows N/P 6, short rods. Scale bar = 2 μιη; and FIG. 3D shows N/P 7, short rods. Scale bar = 1 μιη;

FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D, FIG. 4E, and FIG. 4F show a protein blot analysis of BACE1 : FIG. 4A shows blot protein analysis after infusing worm- shaped nanoparticles with siRNA targeting BACEl(B) or an unrelated sequence (A), over a 7-day period in the right lateral ventricle. Control was infused with 5% glucose solution over a 2-day period; FIG. 4B shows quantification of protein blot analysis from FIG. 4A, ipsilateral; FIG. 4C shows quantification of protein blot analysis from FIG. 4A, contralateral; FIG. 4D shows protein blot analysis after infusing rod (4%) and spherically (8%) shaped nanoparticles, with siRNA targeting BACE1, over a 7-day period in the right lateral ventricle. Control was infused with 5% glucose solution over a 7-day period; FIG. 4E shows quantification of protein blot analysis from FIG. 4D, ipsilateral; and FIG. 4F shows quantification of protein blot analysis from FIG. 4D, contralateral. LFB- left forebrain, RFB- right forebrain, KO - knockout;

FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D, FIG. 5E, and FIG. 5F show a protein blot analysis of BACE1 after infusing worm-shaped nanoparticles: FIG. 5 A shows protein blot analysis with siRNA targeting BACEl(B) or an unrelated sequence (A), over a 7-day period in the right lateral ventricle; FIG. 5B shows quantification of protein blot analysis from FIG. 5A, cervical; FIG. 5C shows quantification of protein blot analysis from FIG. 5A, brainstem; FIG. 5D shows protein blot analysis with siRNA targeting BACE1 , over a 7-day period in the right lateral ventricle; FIG. 5E shows quantification of protein blot analysis from FIG. 5D, thoracic; and FIG. 5F shows quantification of protein blot analysis from FIG. 5D, lumbar. Control was infused with 5% glucose solution over a 2-day period, KO - knockout;

FIG. 6A, FIG. 6B, FIG. 6C, FIG. 6D, FIG. 6E, FIG. 6F, and FIG. 6G show protein blot analysis of BACE1 after infusing rod (4%) and spherically (8%) shaped nanoparticles, with siRNA targeting BACE1, over a 7-day period in the right lateral ventricle: FIG. 6A shows brainstem and thoracic; FIG. 6B shows cervical; FIG. 6C shows lumbar; FIG. 6D shows quantification of protein blot analysis from FIG. 6A, brainstem samples; FIG. 6E shows quantification of protein blot analysis from FIG. 6A, thoracic samples; FIG. 6F shows quantification of protein blot analysis from FIG. 6B), cervical; and FIG. 6G shows quantification of protein blot analysis from FIG. 6C, lumbar. Control was infused with 5% glucose solution over a 7-day period. KO - knockout;

FIG. 7A, FIG. 7B, FIG. 7C, and FIG. 7D show tuning the shape of LPEI-g-

PEG/siRNA micellar nanoparticles by varying PEG grafting degree. FIG. 7A shows LPEI 17k-g-0.6%PEG10k/siRNA nanoparticles (N/P = 5) showing worm-like morphology. FIG. 7B shows LPEI17k-g-0.8%PEG10k/siRNA nanoparticles (N/P = 5) showing rod-like morphology. FIG. 7C shows LPEI17k-g-1.2%PEG10k/siRNA nanoparticles (N/P = 5) showing spherical morphology. All scale bars = 200 nm. FIG. 7D shows particle size measured by dynamic light scattering in water and 150 mmol/1 NaCl after incubation at room temperature for 4 hours (n > 3, mean ± SD). The LPEI17k/siRNA nanoparticles without PEG grafts (0%) were prepared at an N/P ratio of 10 without crosslinking;

FIG. 8A and FIG. 8B show transmission electron microscopy (TEM) images of non-PEGylated crosslinked LPEInk/siRNA particles at N/P ratio of 5, indicating severe aggregation of complexes in 150 mM NaCl. FIG. 8A and FIG. 8B depict two different magnifications of the crosslinked samples as analyzed by TEM with scale bars of 400 nm and 2 μιη, respectively;

FIG. 9A, FIG. 9B, FIG. 9C, and FIG. 9D show TEM images showing the effect of N/P ratio on the shape of LPEIi_7k-g-0.6%PEGi₀k/siRNA micellar nanoparticles. LPEIi7k-g-0.6%PEGiok/siRNA nanoparticles can be varied from long worms to short rods by adjusting the N/P ratio from 3 to 6. Scale bars = 200 nm; FIG. 10A, FIG. 10B, FIG. IOC, FIG. 10D, and FIG. 10E show gel retardation and zeta potential analyses of LPEIn_k-g-PEGio_k/siRNA micellar nanoparticles, and TEM image of LPEIn_k/siRNA nanoparticles. FIG. 10A shows the release of free siRNA from LPEIn_k-g-PEGio_k/siRNA nanoparticles in the presence of 20 μΜ DS. DS-Dextran Sulfate, FS-Free siRNA. FIG. 10B shows the release of siRNA from the same particles when challenged with 150 μΜ DS. FIG. IOC shows TEM analysis of nanoparticles formed by the complexation of LPEIn_k with siRNA (N/P = 10). Scale bar = 100 nm. FIG. 10D and FIG. 10E show zeta potential measurements of nanoparticles encapsulating siRNA formed using LPEInk-g-PEGiok and the non- PEGylated LPEI_17k in water (FIG. 10D) and PBS (FIG. 10E) (n = 3, Mean ± SD, ANOVA, n.s.-between the negatively charged complexes);

FIG. 1 1A, FIG. 1 IB, FIG. 1 1C, FIG. 1 ID, FIG. 1 IE, and FIG. 1 IF show in vitro knockdown efficiency of LPEI17k/siRNA nanoparticles in N2a cells. FIG. 11A shows protein blot analysis oiBACEl and APP levels after N2a cells were transfected with nanoparticles prepared with sequences BACE33 and APP35, respectively, or with Lipofectamine (positive control) and naked sequences (negative control).

Lipofect: Lipofectamine. FIG. 1 IB and FIG. 1 1C show quantification of protein blot analysis oiBACEl (FIG. 1 IB) and APP (FIG. 11C) protein levels as compared to nontransfected N2a cells (n = 4, mean ± SEM, analysis of variance (ANOVA), F = 19.75, P < 0.0001). All in vitro studies were performed at an N/P ratio of 10. FIG. 1 ID show microscopic analysis of the in vitro cell culture model confirmed that fluorescently labeled siRNA (red) was delivered to the cytoplasm of cells using LPEI17K (nuclei-DAPI). The classical pattern of siRNA accumulation in the cell around the nucleus was noted (stained blue). FIG. 1 IE show MTT assay analysis which confirms that over the transfection period of 24 hours, the various formulations built on the LP Ell 7k platform were not toxic (n = 5, mean ± SEM, ANOVA, n.s., not significant). FIG. 1 IF shows transfection studies in N2a cells with varying amounts of siRNA delivered in the form of nanoparticles with the LPEI17k. The first three lanes are a dilution series of protein lysates from untransfected cells;

FIG. 12A, FIG. 12B, FIG. 12C, FIG. 12D, FIG. 12E, and FIG. 12F show the infusion setup and immunohistochemical (IHC) analysis for astrocytic and microglial activation in the mouse hippocampus infused with micellar nanoparticles. FIG. 12A shows an infusion setup allowing for continuous infusion into the lateral ventricle of the brain of a mouse. The tube from the cannula was connected to a slow infusion pump. The animals were awake, freely moving, and had free access to food pellets and water. FIG. 12B shows a schematic of infusion regimen. All infusions were performed at 0.1 μΐ/minute. FIG. 12C and FIG. 12D show IHC staining of tissue sections of the right (ipsilateral) hippocampus showing no significant difference in local recruitment of GFAP+ astrocytes to vehicle (FIG. 12C) and s iRNA/LPEI 17k-g- 0.8%PEG10k nanoparticles (FIG. 12D) after the 7-day infusion protocol. FIG. 12E and FIG. 12F show that similarly, there was no significant difference in microglial cell (Iba-1+) activation following the 7-day infusion protocol for vehicle (FIG. 12E) and siRNA/LPEI17k-g-0.8%PEG10k nanoparticles (FIG. 12F). Images of other sections are in FIG. 16. Scale bar = 200 μιη. Inset scale bar = 20 μιη;

FIG. 13A, FIG. 13B, and FIG. 13C show protein blot analyses detailing examples of initial studies involving siRNA nanoparticles being infused into the brain of mice. All infusions were performed in the right (ipsilateral) lateral ventricle at a dose of 16 μg of siRNA, complexed in nanoparticle form, per day, with the same dosing regimen (shown in FIG. 13B). FB-Forebrain, NP -Nanoparticles, H-

Hippocampus. FIG. 13A, FIG. 13B, and FIG. 13C show protein blot analyses of BACE1 in the forebrain and hippocampus, following infusion with uncrosslinked LPEIi_7k/siRNA nanoparticles (FIG. 13A), crosslinked LPEIn_k/siRNA nanoparticles (FIG. 13B), uncrosslinked LPEIi_7k-g-0.6%PEGio_k/siRNA micellar nanoparticles (FIG. 13C) in the lateral ventricle of the brain;

FIG. 14 shows microscopic analysis of fluorescently labeled siRNA encapsulated in micellar nanoparticles in the brain parenchyma. Animals were infused with micellar nanoparticles in the lateral ventricle for two days prior to harvesting the brain tissue. Fluorescently labeled siRNA (red) is observed in the brain parenchyma, proximal to the lateral ventricle infusion site. DAPI (nuclei) and neurons (green). Scale bar = 20 μιη;

FIG. 15A, FIG. 15B, FIG. 15C, FIG. 15D, FIG. 15E, and FIG. 15F show microscopic analysis of astrocytic cells (GFAP+ staining) in response to brain infusion over 2 days. FIG. 15A, FIG. 15B, and FIG. 15C show vehicle infusion, FIG. 15D, FIG. 15E, and FIG. 15F show nanoparticle infusion (16 μg of encapsulated siRNA/day). FIG. 15A and FIG. 15D show activated glial cells migrating towards the site of injury proximal to the lateral ventricle infusion site. FIG. 15B and FIG. 15E show the gradient in glial cell activation and migration towards the site of injury. FIG. 15C and FIG. 15F show glial cell activation moving farther away from the site of infusion. Scale bar = 20 μιη in (d), for the remaining panels 100 μιη;

FIG. 16A, FIG. 16B, FIG. 16C, FIG. 16D, FIG. 16E, FIG. 16F, FIG. 16G, FIG. 16H, FIG. 161, FIG. 16J, FIG. 16K, FIG. 16L, FIG. 16M, and FIG. 16N show microscopic analysis of astrocytic and microglial activation in the hippocampus as assessed by immunohistochemistry after infusion of micellar nanoparticles containing 64 μg of siRNA following a 7-day infusion protocol. FIG. 16A, FIG. 16B, FIG. 16C, and FIG. 16D show that no significant difference was observed in astrocytic (GFAP staining) response in the contralateral hippocampus when infused with vehicle (FIG. 16A), worm-like nanoparticles (FIG. 16B), spherical nanoparticles (FIG. 16C), and rod-like nanoparticles (FIG. 16D). FIG. 16E, FIG. 16F, FIG. 16G, and FIG. 16H show that no significant difference was observed in microglial (Iba- 1 staining) response in the contralateral hippocampus when infused with vehicle (FIG. 16E), worm-like nanoparticles (FIG. 16F), spherical nanoparticles (FIG. 16G), and rod-like nanoparticles (FIG. 16H). FIG. 161, FIG. 16J, and FIG. 16K show that no significant difference was observed in astrocytic (GFAP staining) response in the ipsilateral hippocampus when infused with vehicle (FIG. 161), worm-like nanoparticles (FIG. 16J), and spherical nanoparticles (FIG. 16K). FIG. 16L, FIG. 16M, and FIG. 16N show that no significant difference was observed in microglial (Iba- 1 staining) response in the ipsilateral hippocampus when infused with to vehicle (FIG. 16L), worm-like nanoparticles (FIG. 16M), and spherical nanoparticles (FIG. 16N). Scale bar = 200 μιη; Inset scale bar = 20 μιη;

FIG. 17A, FIG. 17B, FIG. 17C, FIG. 17D, FIG. 17E, and FIG. 17F show in vivo BACEl knockdown in the cortex and hippocampi of mice infused with worm- like, rod-like, and spherical micellar nanoparticles. All infusions were performed in the right (ipsilateral) lateral ventricle at a dose of 16 μg of siRNA/day with the same dosing regimen for all nanoparticles as show in FIG. 12B (n = 4 for infusion of wormlike, rod-like, and spherically shaped nanoparticles, scrambled and naked siRNA infusion studies were performed in duplicate). FIG. 17A and FIG. 17B show protein blot analysis of BACEl levels in the cortex (FIG. 17A) and hippocampus (FIG. 17B), in both the right (ipsilateral) and left (contralateral) hemispheres after delivery of sequence BACE33 using worm-like (W), spherical (S), and rod-like (R)

nanoparticles. KO, BACE knockout; Co, vehicle infusion; Sc, Scrambled siRNA complexed with LPEI17k-g-0.8%PEG10k; N, naked siRNA sequence BACE33. FIG. 17C, FIG. 17D, FIG. 17E, and FIG. 17F show quantification of BACEl levels from protein blot analysis in the ipsilateral cortex (FIG. 17C) (mean ± SEM, analysis of variance (ANOVA), F = 9.133, P < 0.05, Tukey's multiple comparisons test), contralateral cortex (FIG. 17D) (mean ± SEM, ANOVA, F = 17.91, P < 0.0001, Tukey's multiple comparisons test), ipsilateral hippocampus (FIG. 17E) (mean ± SEM, ANOVA, F = 13.45, P < 0.0001, Tukey's multiple comparisons test), and contralateral hippocampus (FIG. 17F) (mean ± SEM, ANOVA, F = 14.04, P < 0.0001, Tukey's multiple comparisons test); and

FIG. 18A, FIG. 18B, FIG. 18C, FIG. 18D, FIG. 18E, and FIG. 18 F show in vivo BACEl knockdown in the brainstem, and cervical, thoracic, and lumbar sections of the spinal cord of mice, using worm- and rod-like, and spherical micellar nanoparticles. All infusions were performed in the right (ipsilateral) lateral ventricle at a dose of 16 μg of siRNA/day delivered using nanoparticles with the same dosing regimen as shown in FIG. 12B (n = 4 for infusion of worm-like, rod-like, and spherically shaped nanoparticles, whereas scrambled and naked siRNA infusions were performed in duplicate). Protein blot analysis of BACEl levels in the brainstem and cervical section (FIG. 18A), and in the thoracic and lumbar sections of the spinal cord (FIG. 18B) after delivery of sequence BACE33 using worm-like (W), spherical (S) and rod-like (R) micellar nanoparticles. KO: BACEl knockout, Co: vehicle infusion, Sc: Scrambled siRNA complexed with LPEI_17k-g-0.8%PEGi₀k, N: naked siRNA sequence BACE33. FIG. 18 C, FIG. 18D, FIG. 18E, and FIG. 18F show

quantification of BACEl levels from protein blot analysis in the brainstem (FIG. 18C) (Mean ± SEM, ANOVA, F = 24.83, p < 0.0001, Tukey's multiple comparisons test), cervical section of spinal cord (FIG. 18D) (Mean ± SEM, ANOVA, F = 4.762, p < 0.05, Tukey's multiple comparisons test), thoracic section of spinal cord (FIG. 18E) (Mean ± SEM, ANOVA, F = 5.686, p < 0.05, Tukey's multiple comparisons test), and lumbar section of spinal cord (FIG. 18F) (Mean ± SEM, ANOVA, F = 34.35, p < 0.0001, Tukey's multiple comparisons test). The patent or application file contains at least one drawing executed in color.

Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee. DETAILED DESCRIPTION

The presently disclosed subject matter now will be described more fully hereinafter with reference to the accompanying Figures, in which some, but not all embodiments of the inventions are shown. Like numbers refer to like elements throughout. The presently disclosed subject matter may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Indeed, many modifications and other embodiments of the presently disclosed subject matter set forth herein will come to mind to one skilled in the art to which the presently disclosed subject matter pertains having the benefit of the teachings presented in the foregoing descriptions and the associated Figures. Therefore, it is to be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims.

I. METHODS FOR PREPARING SHAPE-CONTROLLED NUCLEIC ACID PARTICLES

The presently disclosed subject matter provides methods for assembling nucleic acid molecules into micellar nanoparticles having tuneable, uniform, and distinct shapes, including worm-like, rod-like, or spherical shapes, using block copolymers or graft copolymers comprising a water soluble, non-charged polymer, such as polyethylene glycol (PEG), and a polycation. In some embodiments, the presently disclosed subject matter provides methods for assembling double-stranded RNA molecules into micellar nanoparticles having tuneable, uniform, and distinct shapes, including worm-like, rod-like, or spherical shapes, using block copolymers or graft copolymers comprising a water soluble, non-charged polymer, such as polyethylene glycol (PEG), and a polycation. By optimizing the copolymer structure and controlling the assembly conditions, the shape of nucleic acid-containing nanoparticles can be tuned from spherical to rod-like or worm-like morphologies. In some embodiments, by optimizing the copolymer structure and controlling the assembly conditions, the shape of siRNA-containing nanoparticles can be tuned from spherical to rod-like or worm-like morphologies. The presently disclosed

nanoparticles can be used to deliver nucleic acids, such as siRNAs, in vivo. Accordingly, in some embodiments, the presently disclosed subject matter provides a method for preparing a shape-controlled nucleic acid nanoparticle, the method comprising: providing a copolymer solution comprising a block copolymer or a graft copolymer of a water soluble, non-charged polymer and a polycation in a first solvent, wherein the water soluble, non-charged polymer and the polycation each have a structure and a molecular weight; and mixing the copolymer solution with a solution of nucleic acid at a predetermined nucleic acid to copolymer ratio and pH in a second solvent to form a shape-controlled nucleic acid nanoparticle, wherein the nucleic acid has a predetermined number of base pairs and the first and second solvent can be the same or different; and wherein the shape-controlled nucleic acid nanoparticle has a shape controlled by one or more of the: structure of the water soluble, non-charged polymer; molecular weight of the water soluble, non-charged polymer; structure of the polycation; molecular weight of the polycation; number of base pairs of the nucleic acid; ratio of nucleic acid to copolymer; pH; first solvent comprising the copolymer solution and/or the second solvent comprising the nucleic acid solution, and, if the copolymer is a graft copolymer, a graft density thereof.

In some embodiments, the presently disclosed subject matter provides a method for preparing a shape-controlled siRNA nanoparticle, the method comprising: providing a copolymer solution comprising a block copolymer or a graft copolymer of a water soluble, non-charged polymer and a polycation in a first solvent, wherein the water soluble, non-charged polymer and the polycation each have a structure and a molecular weight; and mixing the copolymer solution with a solution of siRNA at a predetermined siRNA to copolymer ratio and pH in a second solvent to form a shape- controlled siRNA nanoparticle, wherein the siRNA has a predetermined number of base pairs and the first and second solvent can be the same or different; and wherein the shape-controlled siRNA nanoparticle has a shape controlled by one or more of the: structure of the water soluble, non-charged polymer; molecular weight of the water soluble, non-charged polymer; structure of the polycation; molecular weight of the polycation; number of base pairs of the siRNA; ratio of siRNA to copolymer; pH; first solvent comprising the copolymer solution and/or the second solvent comprising the siRNA solution, and, if the copolymer is a graft copolymer, a graft density thereof.

In some embodiments, the nucleic acid in the shape-controlled nucleic acid nanoparticle is designed to interfere selectively with the transcription, translation and/or expression of a specific polypeptide or protein normally expressed within a cell. A nucleic acid can be RNA or DNA and can be single or double stranded. Examples of nucleic acids that can be used in the presently disclosed subject matter include, but are not limited to, nucleic acid sequence encoding proteins, for example that act as transcriptional repressors, antisense molecules, ribozymes, small inhibitory nucleic acid sequences, for example but are not limited to small interfering RNA (siRNA), a short hairpin RNA or small hairpin RNA (shRNA), a microRNA

(miRNA), antisense nucleotides, aptamers, CRISPR RNAs (e.g., guide RNA) etc. In some embodiments, the nucleic acid is selected from at least one member of the group consisting of a guide RNA, siRNA, miRNA, shRNA, antisense RNA, antisense oligonucleotide, ribozyme, CRISPR RNA, and an aptamer. In some embodiments, the nucleic acid comprises siRNA. In some embodiments, the siRNA is at least 19 base pairs in length. In some embodiments the siRNA is from 19 base pairs to 25 base pairs in length. In some embodiments, the siRNA is 19 base pairs in length. In some embodiments, the siRNA is 20 base pairs in length. In some embodiments, the siRNA is 21base pairs in length. In some embodiments, the siRNA is 22 base pairs in length. In some embodiments, the siRNA is 23 base pairs in length. In some embodiments, the siRNA is 24 base pairs in length. In some embodiments, the siRNA is 25 base pairs in length. In some embodiments, the siRNA comprises about 25 base pairs.

As used herein, a "siRNA" is a double stranded RNA (dsRNA) that interferes with the expression of specific genes with complementary nucleotide sequences. As used herein, a "shRNA" is an artificial dsRNA molecule with a tight hairpin turn. As used herein, a "miRNA" is a small non-coding dsRNA molecule which also functions in RNA silencing. As used herein, an "antisense RNA" (asRNA) is a single-stranded RNA that is complementary to a messenger RNA (mRNA) strand transcribed within a cell. Antisense RNA may be introduced into a cell to inhibit translation of a complementary mRNA by base pairing to it and physically obstructing the translation machinery. As used herein, a "ribozyme" is a catalytic RNA molecule (RNA enzyme) that has a separate catalytic and substrate binding domain. As used herein, an "aptamer" is a nucleic acid that has been engineered through repeated rounds of in vitro selection to bind to various molecular targets such as small molecules, proteins, nucleic acids, and even cells, tissues and organisms. As used herein, an "antisense nucleic acid" is preferably an oligonucleotide, such as a synthetic oligonucleotide, and may comprise deoxyribonucleotides, modified deoxyribonucleotides, or some combination of both. As used herein, a "CRISPR R A" is part of the CRISPR (clustered regularly interspaced palindromic repeats) pathway which provides a complementary approach to RNA interference by regulating gene expression primarily on the transcriptional level.

A "gene," as used herein, refers to a polynucleotide containing at least one open reading frame that is capable of encoding a particular protein after being transcribed and translated. As used herein, a "gene product" is the biochemical material, either RNA or protein, resulting from expression of a gene. A measurement of the amount of gene product is sometimes used to infer how active a gene is. As used herein, "gene expression" is the process by which information from a gene is used in the synthesis of a functional gene product.

As used herein, "double-stranded RNA" (dsR A) or a "double-stranded RNA molecule" is RNA with two complementary strands. As used herein, "small interfering RNA" (siRNA), sometimes known as short interfering RNA or silencing RNA, is a class of double-stranded RNA molecules, usually 19-25 base pairs in length. siRNA interferes with the expression of specific genes with complementary nucleotide sequences in some cases by causing gene silencing or a reduction in gene expression. As used herein, "single-stranded RNA" (ssRNA) or a "single-stranded RNA molecule" is RNA with only one strand.

As used herein, "gene silencing" is a general term that refers to the ability to prevent the expression of a certain gene. As used herein, "gene knockdown" refers to the reduction in expression of one or more genes. The reduction can occur either through genetic modification or by treatment with a reagent such as a short DNA or RNA molecule that has a sequence complementary to either the gene or mRNA transcript produced from the gene. By "reduce" or "reduction", it is meant a decrease in a parameter (e.g., gene expression) as detected by standard art known methods, such as those described herein. As used herein, reduce includes at least a 10% change, at least a 20% change, at least a 30% change, at least a 40% change, at least a 50% change, at least a 60% change, at least a 70% change, at least an 80% change, and in some embodiments, at least a 90% change. In some embodiments, the reduction in gene expression is a complete inhibition of gene expression, such as up to a 100% reduction. In some embodiments, gene knockdown causes a reduction or decrease of gene expression of about 30% to about 50%. As used herein, the term "nucleic acid", "polynucleotide" or "oligonucleotide" refers to a polymer of nucleotides. Typically, a polynucleotide comprises at least three nucleotides. The polymer may include natural nucleosides (i.e., adenosine, thymidine, guanosine, cytidine, uridine, deoxy adenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), nucleoside analogs (e.g., 2-aminoadenosine, 2- thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, C5-propynylcytidine, C5-propynyluridine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5- methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8- oxoguanosine, 0(6)-methylguanine, and 2-thiocytidine), chemically modified bases, biologically modified bases (e.g., methylated bases), intercalated bases, modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose), or modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoramidite linkages).

As used herein, a "peptide" or "protein" comprises a string of at least three amino acids linked together by peptide bonds. The terms "protein" and "peptide" may be used interchangeably. Peptide may refer to an individual peptide or a collection of peptides. Inventive peptides preferably contain only natural amino acids, although non-natural amino acids (i.e., compounds that do not occur in nature but that can be incorporated into a polypeptide chain) and/or amino acid analogs as are known in the art may alternatively be employed. Also, one or more of the amino acids in an inventive peptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc. In a preferred embodiment, the modifications of the peptide lead to a more stable peptide (e.g., greater half-life in vivo). These modifications may include cyclization of the peptide, the incorporation of D-amino acids, etc. None of the modifications should substantially interfere with the desired biological activity of the peptide.

In certain embodiments, the water soluble, non-charged polymer comprises polyethylene glycol (PEG). In particular embodiments, the PEG has a molecular weight ranging from about 500 Da to about 20 kDa. In yet more particular embodiments, the PEG has a molecular weight of about 10 kDa, i.e., PEGio_k- Changing the molecular weight of PEG, for example, in the range of about 500 Da to about 20 kDa, can influence the condensation capacity and nanoparticle shape. It also has been found that PEG chain length and grafting density are important for preparing shape-controlled RNA particles. Too high of a grafting density results in poor complexation and yields no concrete particles. In some embodiments, the grafting density of the PEG ranges from about 0.1% to about 20%. In some embodiments, the copolymer is a graft polymer and the PEG has a graft density ranging from about 0.1% to about 20%. Further, in the absence of PEG on the LPEI backbone (0% grafting density), the particles tend to aggregate and form irregular shaped agglomerates.

In particular embodiments, the PEG has a graft density selected from the group consisting of a 2% graft density, a 4% graft density, and an 8% graft density. In some embodiments, the 2% graft density of the PEG results in a worm-shaped nucleic acid nanoparticle, the 4% graft density of the PEG results in a rod-shaped nucleic acid nanoparticle, and the 8% graft density of PEG results in a spherically- shaped nucleic acid nanoparticle. In some embodiments, the 2% graft density of the PEG results in a worm-shaped siRNA nanoparticle, the 4% graft density of the PEG results in a rod-shaped siRNA nanoparticle, and the 8% graft density of PEG results in a spherically-shaped siRNA nanoparticle. In some embodiments, the shape of the nucleic acid nanoparticle is selected from the group consisting of worm-shaped, spherically-shaped, and rod-shaped. In some embodiments, the shape of the siRNA nanoparticle is selected from the group consisting of worm-shaped, spherically- shaped, and rod-shaped.

In some embodiments, the designed 4% graft density is an actual graft density of 0.8%. In some embodiments, the designed 2% graft density is an actual graft density of 0.6%. As used herein, a "designed graft density" is calculated from the feeding ratio of PEG and the polycation under the assumption that the reaction efficiency is 100%. As used herein, an "actual graft density" is defined as the graft density found after the reaction occurs.

In general, the polycation should have a high charge density so it can be used for nucleic acid, such as siRNA, condensation. In particular embodiments, the polycation is selected from the group consisting of linear polyethylenimine (LPEI), poly-lysine, poly-arginine, poly-histidine, chitosan, branched PEI, a poly (beta- aminoester), a polyphosphoester, polyphosphoramidate (PPA), and PEG-&- polyphosphoramidate (PEG-PPA). In even more particular embodiments, the polycation is LPEI. In some embodiments, the LPEI has a molecular weight ranging from about 2 kDa to about 50 kDa. In certain embodiments, the LPEI has a molecular weight of about 17 kDa, i.e., LPEIn_k.

Typically, in methods known in the art, the nucleic acid, such as siRNA, used in knockdown studies, as well as in nanoparticle formation and delivery applications, has been 19 base pairs (bp) long with 2-bp overhangs. In contrast, the siRNA used herein was 25 bp with blunt ends. Varying the length and types of ends of the base pairs of the nucleic acid, such as the siRNA, can be a determinant in the context of the kind of complexes it can form with polymers. Accordingly, different lengths of nucleic acid, such as siRNA, are envisioned for the presently disclosed methods. .

In addition, the nucleic acid targeting protein of interest can be varied, which allows for developing therapeutics of choice against relevant targets. In particular embodiments, the nucleic acid, such as siRNA, targets the BACE1 gene (e.g., Entrez Gene ID 23621) encoding for β-site APP cleaving enzyme 1, a key enzyme required for the generation of AB from the amyloid-B precursor protein (APP). In yet other embodiments, the nucleic acid, such as siRNA, targets the APP gene (e.g., Entrez

Gene ID 351). In some embodiments, the nucleic acid decreases the expression of B- site APP cleaving enzyme 1 (BACE1) and/or amyloid-B precursor protein (APP). In some embodiments, the siRNA decreases the expression of B-site APP cleaving enzyme 1 (BACE1) and/or amyloid-B precursor protein (APP). In some

embodiments, the siRNA comprises an siRNA sequence that is similar to SEQ ID NO: 1, 2, 3, 4, 5, and 6 as provided in Table 1 herein below. In some embodiments, the siRNA comprises a functional variant and/or fragment of SEQ ID NO: 1, 2, 3, 4, 5, and 6. In some embodiments, the nucleic acid comprises an siRNA sequence that is at least 92% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. In some embodiments, the siRNA comprises an siRNA sequence selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, and 6 as provided in Table 1 herein below. In some embodiments, the siRNA comprises SEQ ID NO: 1. In some embodiments, the siRNA comprises SEQ ID NO: 2. In some embodiments, the siRNA comprises SEQ ID NO: 3. In some embodiments, the siRNA comprises SEQ ID NO: 4. In some embodiments, the siRNA comprises SEQ ID NO: 5. In some embodiments, the siRNA comprises SEQ ID NO: 6.

"Functional variants" of SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6 include siRNA which have at least one property, activity and/or function characteristic of SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6, such as the ability to mediate gene silencing. Generally, fragments or portions of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6 encompassed by the presently disclosed subject matter include those having a deletion (i.e. one or more deletions) of a base pair relative to SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6. Fragments or portions in which only contiguous base pairs have been deleted or in which non-contiguous base pairs have been deleted are also envisioned.

Generally, the siRNA or functional variant thereof has a nucleic acid sequence which is at least about 80% identical, at least about 84% identical, at least about 88% identical, at least about 92% identical, at least about 96% identical, or at least about 100% identical to SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6 over the length of the variant.

The ratio of copolymer nitrogen to nucleic acid phosphate (N/P ratio), such as the ratio of copolymer nitrogen to double-stranded RNA phosphate, also plays an important role in determining the shape of the particles. In representative embodiments, the N/P ratio can range from about 0.1 to about 20. In some embodiments, the ratio of nucleic acid to copolymer is measured as copolymer nitrogen to nucleic acid phosphate (N/P ratio) and has a range from about 0.1 to about 20. In some embodiments, the ratio of siRNA to copolymer is measured as copolymer nitrogen to siRNA phosphate (N/P ratio) and has a range from about 0.1 to about 20. In particular embodiments, the N/P ratio is less than about 10 to mitigate potential cytotoxic effects.

In some embodiments, the first and/or second solvent is water or in a mixture comprising water and a water-miscible solvent selected from the group consisting of dimethylformamide (DMF), dimethyl sulfoxide (DMSO), dioxane, and

tetrahydrofuran (THF). In particular embodiments, the water-miscible solvent is 70% DMF.

In certain embodiments, the pH has a range from about 1 to about 7.5.

In some embodiments, the method further comprises removing the water- miscible solvent from the nucleic acid nanoparticle. In further embodiments, the method further comprises removing the water-miscible solvent from the siRNA nanoparticle. In some embodiments, the method further comprises crosslinking the nucleic acid nanoparticle. In some embodiments, the method further comprises crosslinking the siRNA nanoparticle. Preferably, the crosslinks should be bioreducible or degradable, and, in certain embodiments, reversible. Representative bioreducible or degradable linkages include, but are not limited to:

O O O

M S 11 II II Enzymatic

s - ^ Degradation

ϋ _H

ester disulfide amide anhydride

II. SHAPE-CONTROLLED NUCLEIC ACID PARTICLES

In some embodiments, the presently disclosed subject matter provides a nucleic acid nanoparticle prepared by the presently disclosed methods. In some embodiments, an siRNA nanoparticle prepared by the method described immediately hereinabove is provided.

In some embodiments, the nucleic acid is selected from at least one member of the group consisting of a guide RNA, siRNA, miRNA, shRNA, antisense RNA, antisense oligonucleotide, ribozyme, CRISPR RNA, and an aptamer. In some embodiments, the nucleic acid comprises siRNA.

As used herein the term "monomer" refers to a molecule that can undergo polymerization, thereby contributing constitutional units to the essential structure of a macromolecule or polymer.

As used herein, an "oligomer" includes a few monomer units, for example, in contrast to a polymer that potentially can comprise an unlimited number of monomers. Dimers, trimers, and tetramers are non-limiting examples of oligomers.

A "polymer" is a molecule of high relative molecule mass, the structure of which essentially comprises the multiple repetition of unit derived from molecules of low relative molecular mass, i.e., a monomer.

A "block copolymer" is a copolymer that comprises two or more

homopolymer subunits linked by covalent bonds. The union of the homopolymer subunits may require an intermediate non-repeating subunit, known as a junction block. Block copolymers with two or three distinct blocks are called diblock copolymers and triblock copolymers, respectively.

A "branched copolymer" consists of a single main chain with one or more polymeric side chains. A "graft copolymer" is a branched copolymer in which the side chains are structurally, either constitutionally or configurationally, distinct from the main chain.

Further, as used herein, the term "nanoparticle," refers to a particle having at least one dimension in the range of about 1 nm to about 1000 nm, including any integer value between 1 nm and 1000 nm (including about 1, 2, 5, 10, 20, 50, 60, 70, 80, 90, 100, 200, 500, and 1000 nm and all integers and fractional integers in between). In some embodiments, the nanoparticle has at least one dimension, e.g., a diameter, of about 100 nm. In some embodiments, the nanoparticle has a diameter of about 200 nm. In other embodiments, the nanoparticle has a diameter of about 500 nm. In yet other embodiments, the nanoparticle has a diameter of about 1000 nm (1 μηι). In such embodiments, the particle also can be referred to as a "microparticle. Thus, the term "microparticle" includes particles having at least one dimension in the range of about one micrometer (μιη), i.e., 1 x 10^"6 meters, to about 1000 μιη. The term "particle" as used herein is meant to include nanoparticles and microparticles.

It will be appreciated by one of ordinary skill in the art that nanoparticles suitable for use with the presently disclosed methods can exist in a variety of shapes, including, but not limited to, spheroids, rods, disks, pyramids, cubes, cylinders, nanohelixes, nanosprings, nanorings, rod-shaped nanoparticles, arrow-shaped nanoparticles, teardrop-shaped nanoparticles, tetrapod-shaped nanoparticles, prism- shaped nanoparticles, and a plurality of other geometric and non-geometric shapes.

III. METHODS FOR TREATING A DISEASE OR CONDITION BY USING SHAPE-CONTROLLED NUCLEIC ACID NANOPARTICLES

In some embodiments, the presently disclosed subject matter provides a method for treating a disease or condition, the method comprising administering to a subject in need of treatment thereof, a shape-controlled nucleic acid nanoparticle described herein, or a pharmaceutical composition thereof, in an amount effective for treating the disease or condition. In some embodiments, the nucleic acid is selected from at least one member of the group consisting of a gRNA, siRNA, miRNA, shRNA, antisense RNA, antisense oligonucleotide, ribozyme, CRISPR RNA, and an aptamer. In some embodiments, the nucleic acid comprises siRNA. In some embodiments, the presently disclosed subject matter provides a method for treating a disease or condition, the method comprising administering to a subject in need of treatment thereof, a shape-controlled siRNA nanoparticle described immediately hereinabove, or a pharmaceutical composition thereof, in an amount effective for treating the disease or condition.

In some embodiments, the method comprises administering the shape- controlled nucleic acid nanoparticle to the brain and/or spinal cord of the subject. In certain embodiments, the method comprises administering the shape-controlled siRNA nanoparticle to the brain and/or spinal cord of the subject. In particular embodiments, the disease or condition comprises a neurodegenerative disease. In more particular embodiments, the neurodegenerative disease is selected from the group consisting of Amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Parkinson's disease, Alzheimer's disease, Huntington's disease, and dementia with Lewy Bodies.

In some embodiments, the presently disclosed methods further comprise the knockdown of one or more genes. In some embodiments, the nucleic acid decreases the expression of B-site APP cleaving enzyme 1 (BACEl) and/or amyloid-B precursor protein (APP). In some embodiments, the siRNA decreases the expression of B-site APP cleaving enzyme 1 (BACEl) and/or amyloid-B precursor protein (APP). In some embodiments, the nucleic acid comprises an siRNA sequence that is at least 92% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. In some embodiments, the siRNA sequence comprises an siRNA sequence selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, and 6.

The presently disclosed nanoparticles have unique compositions and are reversibly crosslinked to achieve high circulation stability under physiological conditions. Accordingly, the distinct shapes of nanoparticles play a key role towards creating a translatable platform for developing RNA therapeutics. The presently disclosed nanoparticles exhibit lower toxicity as compared to previous formulations and when delivered to the brain and spinal cord can knockdown specific genes of interest in a shape-dependent manner.

Representative uses for the presently disclosed shape-controlled double- stranded RNA particles include intra-ventricular infusion, direct placement in brain parenchyma, intrathecal delivery, intravenous infusion, and direct infusion/injection to the target organ of interest. In some embodiments, different shapes can be optimized for different organs/tissue/cell types. In other embodiments, ligand density on differing shapes can be optimized based on the target of interest. In still other embodiments, the presently disclosed particles can be coupled with triggered release mechanisms.

In some embodiments, the presently disclosed particles do not exhibit aggregation, and are stable in aqueous media and under physiological conditions.

By "disease" is meant any condition, dysfunction or disorder that damages or interferes with the normal function of a cell, tissue, or organ.

The subject treated by the presently disclosed methods in their many embodiments is desirably a human subject, although it is to be understood that the methods described herein are effective with respect to all vertebrate species, which are intended to be included in the term "subject." Accordingly, a "subject" can include a human subject for medical purposes, such as for the treatment of an existing condition or disease or the prophylactic treatment for preventing the onset of a condition or disease, or an animal subject for medical, veterinary purposes, or developmental purposes. Suitable animal subjects include mammals including, but not limited to, primates, e.g., humans, monkeys, apes, and the like; bovines, e.g., cattle, oxen, and the like; ovines, e.g., sheep and the like; caprines, e.g., goats and the like; porcines, e.g., pigs, hogs, and the like; equines, e.g., horses, donkeys, zebras, and the like; felines, including wild and domestic cats; canines, including dogs;

lagomorphs, including rabbits, hares, and the like; and rodents, including mice, rats, and the like. An animal may be a transgenic animal. In some embodiments, the subject is a human including, but not limited to, fetal, neonatal, infant, juvenile, and adult subjects. Further, a "subject" can include a patient afflicted with or suspected of being afflicted with a condition or disease. Thus, the terms "subject" and "patient" are used interchangeably herein.

As used herein, in general, the "effective amount" of an active agent or drug delivery device refers to the amount necessary to elicit the desired biological response. As will be appreciated by those of ordinary skill in this art, the effective amount of an agent or device may vary depending on such factors as the desired biological endpoint, the agent to be delivered, the composition of the encapsulating matrix, the target tissue, and the like. rv. General Definitions

Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this presently described subject matter belongs.

While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter. These definitions are intended to supplement and illustrate, not preclude, the definitions that would be apparent to one of ordinary skill in the art upon review of the present disclosure.

"Biocompatible": The term "biocompatible", as used herein is intended to describe compounds that are not toxic to cells. Compounds are "biocompatible" if their addition to cells in vitro results in less than or equal to 20% cell death, and their administration in vivo does not induce inflammation or other such adverse effects. In some embodiments, the materials used herein are biocompatible.

"Biodegradable": As used herein, "biodegradable" compounds are those that, when introduced into cells, are broken down by the cellular machinery or by hydrolysis into components that the cells can either reuse or dispose of without significant toxic effect on the cells (i.e., fewer than about 20% of the cells are killed when the components are added to cells in vitro). The components preferably do not induce inflammation or other adverse effects in vivo. In certain preferred

embodiments, the chemical reactions relied upon to break down the biodegradable compounds are uncatalyzed.

" Following long-standing patent law convention, the terms "a," "an," and "the" refer to "one or more" when used in this application, including the claims. Thus, for example, reference to "a subject" includes a plurality of subjects, unless the context clearly is to the contrary (e.g., a plurality of subjects), and so forth.

Throughout this specification and the claims, the terms "comprise,"

"comprises," and "comprising" are used in a non-exclusive sense, except where the context requires otherwise. Likewise, the term "include" and its grammatical variants are intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items.

For the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing amounts, sizes, dimensions, proportions, shapes, formulations, parameters, percentages, quantities, characteristics, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term "about" even though the term "about" may not expressly appear with the value, amount or range. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are not and need not be exact, but may be approximate and/or larger or smaller as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art depending on the desired properties sought to be obtained by the presently disclosed subject matter. For example, the term "about," when referring to a value can be meant to encompass variations of, in some embodiments, ± 100% in some embodiments ± 50%, in some embodiments ± 20%, in some embodiments ± 10%, in some embodiments ± 5%, in some embodiments ±1%, in some embodiments ± 0.5%, and in some embodiments ± 0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed compositions.

Further, the term "about" when used in connection with one or more numbers or numerical ranges, should be understood to refer to all such numbers, including all numbers in a range and modifies that range by extending the boundaries above and below the numerical values set forth. The recitation of numerical ranges by endpoints includes all numbers, e.g., whole integers, including fractions thereof, subsumed within that range (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5, as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like) and any range within that range.

EXAMPLES

The following Examples have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill can appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter. The following Examples are offered by way of illustration and not by way of limitation. EXAMPLE 1

Modulating the Shape of siRNA-loaded Nanoparticles into

Worms, Rods, and Spheres

In certain aspects, the presently disclosed subject matter provides a method for preparing siR A micellar nanoparticles using a graft copolymer of polyethylene glycol (PEG) and a polycation, such as linear polyethylenimine (LPEI). Several parameters influence nanoparticle assembly and the shape of nanoparticles. These parameters can include, structure and chain length of the polycation, PEG molecular weight and grafting density, R A sequence and length, RNA to polymer ratio, assembly media and pH, and the like. Using LPEI-g-PEG copolymer as an example, it has been shown that the shape of siRNA-loaded nanoparticles can be controlled to form worm-like, rod-like, or spherical morphologies, within the 100 nm range (FIG. 7A, FIG. 7B, FIG. 7C, FIG. 7D, FIG. 8A, and FIG. 8B). The data suggests that nanoparticle shape might play an important role toward determining delivery efficiency in the nervous system. These shaped siRNA nanoparticles can be stabilized by crosslinking with, for example, disulfide crosslinks.

Particle formulation: LPEInk- x% PEGiok is first modified with Trauts reagent in the presence of dithiothreitol (DTT). Trauts reagent is dissolved in deionized water (cell culture grade) to a concentration of 2 mg/mL. DTT also is dissolved in deionized water to a concentration of 20 mg/mL. 9 μϊ_^ of 50 mM polymer solution is mixed with 18.6 μϊ_^ of Trauts reagent solution and 13 μϊ_^ of the DTT solution. 59.4 of water is added to this mixture and the pH is adjusted to approximately 7.3. The reaction is carried out for 2.5 hours. At the end of the reaction, the mixture is desalted using an Amicon centrifugal filter (3-kDa MWCO) with water 3 times and reconstituted to a volume of 110 μί. 10 of DTT is added to this solution and the pH is adjusted to approximately 1.8 - 2.0. 9.8 μΐ_^ of 100 μΜ siRNA (equivalent to 16 μg of siRNA) is added to 60 μΐ_^ of water. Each batch of siRNA solution is mixed with 60 μϊ_^ of the polymer solution and vortexed. The nanoparticle solution is then dialyzed against water overnight to remove DTT and other reagents. Crosslinking is carried out for two nights with aerial oxidation. The nanoparticle solution is then once again dialyzed for 24 hours against water and then the samples are analyzed by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Worm- shaped nanoparticles are formed using LPEInk- 2% PEGiok, rod-shaped nanoparticles are formed using LPEInk- 4% PEGiok, and spherically shaped nanoparticles are formed using LPEInk- 8% PEGiok-

Particle formulation for observing solvent polarity effect and secondary structure formation: LPEIn_k- 2% PEGio_k is first modified with Trauts reagent in the presence of DTT. Trauts reagent is dissolved in deionized water (cell culture grade) to a concentration of 2 mg/mL. DTT is also dissolved in deionized water to a concentration of 20 mg/mL. 9 μϊ_^ of 50 mM polymer solution is mixed with 18.6 μϊ_^ of Trauts reagent solution and 13 of the DTT solution. 59.4 of water is added to this mixture and the pH is adjusted to approximately 7.3. The reaction is carried out for 2.5 hours. At the end of the reaction, the mixture is desalted using an Amicon centrifugal filter (3-kDa MWCO) with water 3 times and reconstituted to a volume of 110 μΐ_^. 10 μΐ_^ of DTT is added to this solution and the pH is adjusted to

approximately 1.8-2.0. 163.3 μϊ_^ of dimethylformamide (DMF) is added to 60 μϊ_^ of the polymer solution followed by mixing with 9.8 of 100 μΜ siRNA (equivalent to 16 μg of siRNA) which yields a 70% DMF solution. The nanoparticle solution is then dialyzed against 70% DMF overnight to remove DTT and other reagents.

Crosslinking is carried out for two nights with aerial oxidation in the presence of 70% DMF. At the end of two nights, the particles are dialyzed extensively against cell- culture grade water and then further analyzed by dynamic light scattering (DLS) and transmission electron microscopy (TEM).

siRNA: The siRNA utilized in the experiments was 25 base pairs long, with blunt ends, and custom modified (developed by the manufacturer). Examples of siRNA sequences used in the presently disclosed methods are shown in Table 1 (top strand shown of double-stranded siRNA).

In other embodiments, the siRNA sequence shows at least 80% similarity to SEQ ID NO: 1, 2, 3, 4, 5, or 6. In still other embodiments, the siRNA sequence shows at least 90% similarity to SEQ ID NO: 1, 2, 3, 4, 5, or 6.

The term "% similarity" or "percent identity," as known in the art, is a relationship between two or more polypeptide sequences or two or more

polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. "Identity" and "similarity" can be readily calculated by known methods, including but not limited to those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, New Jersey (1994); Sequence Analysis in

Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1991). Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the Megalign program of the

LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences may be performed using the Clustal method of alignment (Higgins and Sharp (1989) CABIOS. 5: 151-153) with the default parameters, including default parameters for pairwise alignments.

The term "sequence analysis software" refers to any computer algorithm or software program that is useful for the analysis of nucleotide or amino acid sequences. "Sequence analysis software" may be commercially available or independently developed. Typical sequence analysis software will include but is not limited to the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wis.), BLASTP, BLASTN, BLASTX (Altschul et al. (1990) J. Mol. Biol. 215:403-410, and DNASTAR (DNASTAR, Inc., Madison, Wis.). Within the context of this application it will be understood that where sequence analysis software is used for analysis, that the results of the analysis will be based on the "default values" of the program referenced, unless otherwise specified. As used herein "default values" will mean any set of values or parameters which originally load with the software when first initialized.

EXAMPLE 2

Characterization and Stability of Worm-Shaped Nanoparticles The presently disclosed subject matter discloses that the shape of siRNA- loaded nanoparticles can be controlled to form worm-like, rod-like, or spherical morphologies, within the 100-nm range (FIG. 7A, FIG. 7B, and FIG. 7C). siRNA micellar nanoparticles were produced using linear polyethylenimine (LPEI) grafted with varying degrees of polyethylene glycol (PEG) as a condensing agent. The shape of the siRNA-loaded nanoparticles could be controlled to form worm-like, rod-like, or spherical morphologies (FIG. 7A, FIG. 7B, FIG. 7C, and FIG. 7D). These particles were made at an N/P ratio of 5, to mitigate cytotoxic effects observed at higher N/P ratios, and stabilized with disulfide crosslinks. They did not exhibit aggregation, and were stable in water and under physiological conditions.

More particularly, FIG. 7A, FIG. 7B, FIG. 7C, and FIG. 7D show that using 2%, 4%, and 8% graft degrees of PEG yields differing shapes of nanoparticles. FIG. 8A and FIG. 8B depict the extensive aggregation of LPEI/siRNA particles after crosslinking and carrying out reactions under a similar protocol to the PEGlyated polymers (FIG. 7A, FIG. 7B, FIG. 7C, and FIG. 7D). These data suggest that the condensation driven by the PEG on the LPEI backbone plays a critical role toward forming distinct species.

To assess the stability of the particles over time, the particles were incubated at room temperature (or 4°C) for 3 months. It was noted that the worm-like particle morphology was still intact after this period of time.

EXAMPLE 3

Solvent Polarity Effect and Formation of Secondary Structure The effect of solvent polarity on the shape characteristics during nanoparticle formation was evaluated. When the initial particle formation conditions were changed from distilled (DI) water to 70% DMF, a difference in the morphology of the nanoparticles was observed (comparing FIG. 7A and FIG. 1A). The switch from DI water to 70% DMF led to the formation of particles with morphology similar to that of short rods as opposed to worms in the previous case. It is important to note that 70% DMF conditions were maintained only during the initial phase of particle formation and crosslinking steps, after which the particles were dialyzed extensively against cell culture grade DI water. After incubating the particles in water for two weeks, TEM was again performed on the dialyzed samples and a worm-like morphology was once again observed (FIG. IB) similar to the case of making particles in purely DI water.

Without wishing to be bound to any one particular theory, this evidence suggests that the worm-like morphology might be a secondary structure that is obtained after the initial particle formation step. Thus, it might be possible to create additional secondary structures that can be formed from simpler primary structures that can be utilized as building blocks.

EXAMPLE 4

Effect of Differing siRNA Sequences

siRNA sequences targeting either β-site APP cleaving enzyme 1 (seqB) or amyloid-β precursor protein (seqA) were selected. As stated above, both sequences were 25 bp long with similar modifications. When nanoparticles were made with seqB using the LPEIn_k- 4% PEGio_k polymer, at N/P 5, short rods were obtained. When the protocol was repeated using seqA, however, worm-like species were obtained once again (FIG. 2A and FIG. 2B). This observation suggests that the sequence of the siRNA species might play a key role towards determining the morphology of the particles, as well. seqB is able to form both worm- and rod-shaped morphologies, whereas utilizing seqA leads to the formation of worm-shaped species independent of using a 2% or a 4% PEGio_k grafted on the LPEIn_k.

EXAMPLE 5

Effect of N/P Ratio on the Shape Characteristics

To understand the sensitivity of the shape modulation protocol on siRNA- loaded nanoparticles, the N/P ratio was varied during the particle formulation step. An example when using the LPEIn_k- 2% PEGio_k system is shown here. The experimental procedures detailed hereinabove were used once again and the ratio of polymer to siRNA mixed in the initial particle formulation step was varied. The range of shapes that can be created by manipulating this ratio can be seen in FIG. 3 A, FIG. 3B, FIG. 3C, and FIG. 3D. Worm-like species are created at N/P 4-5 and short rods are formed at N/P 5-7 as the N/P ratio is increased. There also is evidence of very long worm-shaped species as the N/P ratio is increased. This observation suggests a possible secondary structure being created from the shorter worm-shaped species that can be observed in FIG.3B. Thus, within this polymer subgroup, the significant effect of this parameter can be appreciated.

EXAMPLE 6

Knockdown Effect in the Central Nervous System The effect of different shapes of nanoparticles on their delivery efficiency to the CNS in awake and freely moving non-transgenic mice was evaluated via intraventricular infusion of up to 64 μg of siRNA over a 7-day period. It was shown that LPEI_17k encapsulating siRNA was able to reduce the protein levels of BACEl and APP, in an N2a cell culture model, by 63.3 ± 25.4% and 75.6 ± 10.2%, respectively, thus achieving selective and efficient modulation of these proteins. Using confocal microscopic analysis, it was confirmed that nanoparticles gained access to the cytoplasm of cells near the infusion site. Importantly, whereas only modest knockdown of BACEl levels by worm-like and spherical nanoparticles was observed in the forebrain, a more robust knockdown was achieved by rod-shaped nanoparticles (FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D, FIG. 4E, FIG. 4F). BACEl knockdown also was observed in the hippocampi and the spinal cords of mice infused with nanoparticles (FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D, FIG. 5E, FIG. 5F, FIG. 6A, FIG. 6B, FIG. 6C, FIG. 6D, FIG. 6E, and FIG. 6F). The data suggest that rod-shaped nanoparticles serve as optimal vehicles for the delivery of siRNAs to the CNS.

The presently disclosed subject matter demonstrates the utility of shape- controlled nanoparticles for the delivery of siRNA to knockdown BACEl and APP in in vitro and in vivo models as a potential therapeutic strategy to attenuate AB amyloidosis.

EXAMPLE 7

Intraventricular Delivery of siRNA Nanoparticles to the Central Nervous System Materials and Methods

Synthesis and characterization of LPEI-g-PEG copolymer. Linear polyethyleneimine (LP EI HC1 salt, M_n of LPEI = 17 kDa) was a gift from Polymer Chemistry Innovations (Tucson, AZ). N-Hydroxysuccinimidyl ester of methoxy polyethylene glycol hexanoic acid (PEG-NHS, M_n = 10 kDa) was purchased from NOF America Corporation (White Plains, NY). The LPEI-HCl (7.95 mg, 0.1 mmol of amine) was dissolved in 1 ml of DI water. NaOH solution was added to the solution drop-wise to raise the pH to 6. Then 80 mg of PEG-NHS (designed grafting degree of PEG per amine on LPEI = 8%) was added to the solution and the reaction mixture was vortexed. After incubation overnight, the reaction mixture was dialyzed against DI water and lyophilized to yield a white foam-like solid with a 95% yield. The molecular weight of the graft copolymer was characterized by GPC (gel permeation chromatography) using an Agilent 1200 series Isocratic HPLC System equipped with TSKgel G3000PWxl-CP column and TSKgel G5000PWxl-CP column (Tosoh America, Grove City, OH), which was connected with a multi-angle light scattering detector (MiniDawn, Wyatt Technology, Santa Barbara, CA). The LPEI_17k- g-PEGlOk polymer was obtained with a PEG grafting degree of 1.2%, which corresponds to an average of 4.6 PEG grafts per LPEI molecule. For two other LPEIn_k-g-PEGlOk copolymers with designed grafting degree of 4% and 2%, the actual PEG grafting degrees were 0.8 and 0.6%, respectively.

Particle formulation. LPEInk-g-PEGiok was first modified with Trauts reagent (Sigma, St. Louis, MO) in the presence of dithiothreitol (DTT, Thermo Scientific, Rockford, IL). Trauts reagent was dissolved in deionized water (cell culture grade, Corning, Manassas, VA) to a concentration 2 mg/ml, and DTT was dissolved in DI water to a concentration of 20 mg/ml. An aliquot of 9 μΐ of 50 mmol/1 polymer solution was mixed with 18.6 μΐ of Trauts reagent solution and 13 μΐ of the DTT solution, 59.4 μΐ of water was added to this mixture, and the pH was adjusted to 7.3. At the end of this 2.5-hour reaction, the mixture was desalted using an Amicon centrifugal filter (3 KDa MWCO, Sigma-Aldrich) with water three times and reconstituted to a volume of 1 10 μΐ. An aliquot of 10 μΐ of DTT solution was added to this solution and the pH adjusted to -1.8-2.0. On the other hand, 9.8 μΐ of 100 μιηοΐ/ΐ siRNA (equivalent to 16 μg of siRNA) solution was added to 60 μΐ of water, and mixed with 60 μΐ of the polymer solution and vortexed. The nanoparticle solution was then dialyzed against water overnight to remove DTT and other reagents.

Crosslinking was carried out for 48 hours with aerial oxidation similar to a previous study (Jiang et al. (2013) Adv Mater 25: 227-232). The nanoparticle solution was then dialyzed for 24 hours against water following which the samples were analyzed by dynamic light scattering using a Malvern Zetasizer Nano ZS, which also provided information about zeta potential, followed by TEM analysis for nanoparticle shapes. The protocols detailed above and in the following section were designed to yield nanoparticles with an N/P ratio of 5. The volume of mixing between the polymer and siRNA was scaled accordingly to achieve additional N/P ratios ranging from 3 to 6.

Transmission electron microscopy. An aliquot of 10 μΐ of nanoparticle solution was deposited on ionized nickel grid covered by carbon. The excess liquid on the grid was pipetted out after 7 min, and then 6 μΐ of 2% uranyl acetate solution was deposited on the grid and allowed to incubate for 30 seconds. The excess liquid was once again pipetted out and the grid was allowed to dry at room temperature prior to being examined. The samples were imaged on a Tecnai FEI-12 electron microscope.

Intraventricular infusion in mice. Following anesthetization, the hair above the skull of C57BL/6J mice was removed to expose the scalp. An incision was made along the midline to expose the skull. A hole was drilled through the skull, above the right lateral ventricle (bregma-0.5 mm, 1.0 mm lateral). After drilling, bone fragments were cleaned away. An Alzet apparatus (brain infusion kit# 3, Cupertino, CA) was used as per manufacturer's specifications to place a cannula at a depth of 2.2 mm. The cannula was cemented using dental cement. A sufficiently long tube (FEP- tubing, SCIPRO, Sanborn, NY), so as to allow free head and neck movement of the mice, was used to connect the end of the cannula above the skull to a slow infusion pump (Stoelting, Wood Dale, IL). The animal was then placed in a special enclosure, Raturn Microdialysis Stand-Alone System (with free access to food and water) where the tube going to the slow infusion pump can be secured and the process of infusing the therapeutic agent was begun (0.1 μΐ/minute during the infusion phase). At any given point, there would be only one mouse present in the Raturn Microdialysis Stand-Alone System undergoing infusion (BASi, West Lafayette, IN). A slow infusion pump was used to facilitate the flexibility of having a system that would be able to deliver reagents from a period varying from 2 to 7 days with the ability to stop infusions as per the staggered infusion protocol (FIG. 12B). Importantly, it allowed delivery of a specific volume of therapeutic to the targeted area in the brain and gave the ability to monitor the effect in the live animal, which would mimic a clinical setting where the therapeutic can be potentially used. The right and left hemisphere were referred to, respectively, as the ipsilateral or contralateral side of the brain with reference to the side of infusion.

Immunohistochemical analysis. Mice (controls and those undergoing infusion) were perfused with cold 4% paraformaldehyde in phosphate-buffered saline. The brains were harvested and each hemisphere separated following which they were embedded in paraffin, sectioned sagitally, and processed for immunohistochemical analysis using the peroxidase-antiperoxidase method (Laird et al. (2005) JNeurosci 25: 1 1693-11709) with antibodies specific to BACE1(1 :500), glial fibrillary acidic protein (GFAP, 1 :500, Dako Cytomation, Carpinteria, CA), and Iba-1 (1 :500, Wako Chemicals, Richmond, VA). Secondary, biotinylated, goat-anti-rabbit antibodies were purchased from Vectashield (Burlingame, CA). The sections were counter- stained with hematoxylin.

Western blot analysis. Harvested mouse brains (cortex, hippocampus, and brain stem) and spinal cords (cervical, thoracic, and lumbar) were homogenized with radio-immunoprecipitation buffer (Sigma) in the presence of protease inhibitors (Thermo Scientific, Rockford, IL). Following the manufacturer recommended protocol, lysates were centrifuged at 14,000 rpm at 4 °C for 20 minutes, and the supernatant was used for western blotting. The lysates were run on a 4-12% bis-tris gel (Life Technologies, Grand Island, NY) and then transferred on to a polyvinylidene difluoride (PVDF) membrane. For western blot analysis, the membranes were blocked in 5% milk in Tris buffered saline-Tween 20 - (TBS-T) for an hour and then probed with antibodies specific for BACE1 (1 :2,500), glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) (1 :20,000, Sigma), APP-CTF (1 :8,000), and Actin (1 :5,000, Sigma). Band densitometry analysis was performed using Image Studio Lite software from LI-COR (Lincoln, NE).

In vitro knockdown and cell viability studies. The siRNAs used in this study were BACE33 (SEQ ID NO:2), BACE 23 (SEQ ID NO:3), APP35 (SEQ ID NO: l), APP34 (SEQ ID NO: 6) and APP33 (SEQ ID NO: 5). Luciferase Stealth control and fluorescently labeled (Alexa-555) sequences were all purchased from Life

Technologies. Lipofectamine2000 in Opti-Mem media was used for transfection as per manufacturer recommendations (Life Techonologies). Transfection experiments were performed in a six-well plate with N2a cells maintained in 10% fetal bovine serum (FBS), l x Glutamax, 1 * MEM-NEAA, 1 * sodium pyruvate and antibiotic free conditions (Life Technologies). Cells were transfected with siRNA packaged in nanoparticles or Lipofectamine2000. Cell culture medium was replaced after 16 hours with fresh medium and the cells were harvested for further analysis after 24 hours. For harvesting cells, the media was first aspirated from each well and then each well was washed twice with cold (4 °C) phosphate-buffered saline. A cell scraper was used to collect the cells from each well and protein extraction was performed using the radio-immunoprecipitation buffer following a similar protocol as the tissue protein extraction. The prepared lysates were further probed for protein content via western blot analysis. Cell viability was checked via an 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay based on the manufacturer recommended protocol (Life Technologies). In a 96-well plate, N2A cells were treated at a scale proportional to that of a transfection in a six-well plate. A similar time scale of incubation for 24 hours was maintained after which time knockdown analysis was performed. The cytotoxicity of the samples was analyzed on an EPOCH BIOTEK (Winooski, VT) plate reader.

Confocal fluorescence imaging. Mice (with neurons sparsely expressing GFP,

Thy-1 Promoter, gift from the Richard Huganir lab at Johns Hopkins University) were perfused with cold 4% paraformaldehyde in phosphate-buffered saline 24 hours after undergoing infusion protocol. The brains were harvested, each hemisphere was separated, and they were postfixed 4% paraformaldehyde for 24 hours. The tissues were then treated for 48 hours with 30% sucrose for cryopreservation, placed in a mold with OCT and prepared for cryosectioning. The tissue samples were sectioned sagitally (16-μιη thickness), and collected on superfrost glass and probed with antibodies specific to BACE1 (1 :500), GFAP (Dako Cytomation), and Microglia (Wako Chemicals). Mounting media (Life Technologies) preloaded with DAPI was applied prior to application of the coverslip. Microscopic analyses were performed on a Zeiss LSM 510 microscope. In the case of cell culture, cells were grown on a cover slip placed at the bottom of a six- well plate. At the end of the transfection study with fluorescently labeled siRNA over the same conditions as the knockdown studies, the cover slip was washed in phosphate-buffered saline, and 0.2 M acetic acid in 0.5 M sodium chloride solution. Then, the cover slips were treated with 0.4% Trypan blue solution Hanks buffered salt solution to quench extracellular fluorescence (Rejman et al. (2004) Biochem J 311(Pt 1): 159-169. Mounting media (Life Technologies) preloaded with DAPI was applied to a glass slide prior to placing the cover slip on it for further microscopic analysis. Gel retardation assay. A 2% agarose gel (with ethidium bromide) in a TAE buffer was used to elucidate release of siRNA from micellar nanoparticle

formulations. Nanoparticle formulations were incubated with varying concentrations of dextran sulfate, following which 20 μΐ of each sample was loaded in each well with loading buffer. Samples were run on the gel for 20 minutes at 120 V.

Statistical analysis. Statistical comparisons were performed using ordinary one-way analysis of variance. Comparisons between groups were performed using Tukey's multiple comparisons test using GraphPad Prism Software (La Jolla, CA). Errors bars used in this study were in SD and SEM and noted in the Figures.

Introduction

Alzheimer's disease (AD) is a progressive neurodegenerative disease that significantly impairs memory and cognitive function and affects close to 35 million people worldwide (Holtzman et al. (2011) Sci Transl Med 3: 77sr71; Ridge et al. (2013) PLoS One 8:e79771). Pathological hallmarks of AD include the presence of amyloid plaque deposits and neurofibrillary tangles in the affected brain tissue (Choi et al. (2014) Nature 515: 274-278). The continued formation of amyloid-β (ΑβΙ^ΙΟ and Αβ1^12) species by successive cleavage of the amyloid precursor protein (APP) by β-secretase (BACE1) and γ-secretase leads to the formation of toxic amyloid plaques in the extracellular space (Koffie et al. (2009) Proc Natl Acad Sci USA 106: 4012^1017). While BACE1 knockout mice failed to generate amyloid-β, significant behavioral deficits were observed (Savonenko et al. (2008) Proc Natl Acad Sci USA 105: 5585-5590; Aguzzi and O'Connor (2010) Nat Rev Drug Discov 9: 237-248). Additional genetic studies have shown that moderate reductions in BACE1 and γ- secretase can effectively reduce amyloidosis (Chow et al. (2010) Sci Transl Med 2: 13ral). However, clinical trials using BACE1 and γ-secretase inhibitors to attenuate amyloidosis have been halted recently, due to toxicities and off-targeting effects (Cai et al. (2012) EMBO Mol Med 4: 980-991 ; Imbimbo and Giardina (201 1) Curr Top Med Chem 1 1 : 1555-1570). Thus, there is an urgent need to develop new methods to specifically modulate these targets of interest because of the burgeoning aging population.

RNA interference (RNAi) therapeutics have been actively pursued for selective gene knockdown and currently tested in several clinical trials (Kanasty et al. (2013) Nat Mater 12: 967-977). RNAi offers promise to selectively knockdown the key players implicated in the AD pathway. To fully realize the potential of RNA therapeutics, including short interfering RNAs (siRNAs), effective cationic carriers can be tailored to package these siRNAs into nanoparticles or complexes, which serve to protect the RNA therapeutics and facilitate the delivery and uptake of the nanoparticles into target cells. This siRNA delivery strategy has been particularly successful through intravascular administration, leading to liver-targeted delivery and cancer-targeted delivery where the enhanced permeation and retention effect can be exploited as a means of selective delivery (Lee et al. (2013) Biomed Res Int 2013: 782041 ; Coelho et al. (2013) N Engl J Med 369: 819-829). A recent study showed the use of a gold nanoparticle platform to deliver siRNA to target the antiapototic pathway in glioblastoma multiforme (GBM) in vivo mouse models (Jensen et al. (2013) Sci Transl Med 5: 209ral52). Significant progress has been made to reduce the immunogenicity of viral carriers for the purposes of gene therapy in the central nervous system (CNS) (Gray et al. (2013) Gene Ther 20: 450^159). Here, the focus is on delivering siRNA using a nonviral delivery strategy so as to mitigate the safety concerns associated with viral vectors. Nonviral carriers have been shown to have a good safety profile and have been employed for delivery of siRNA in humans (Lee et al. (2013) Biomed Res Int 2013: 782041; Davis et al. (2010) Na ture 464: 1067-1070). More recently antisense therapy, and the development of single stranded optimized siRNA sequences, has emerged as an alternative method that was shown to knockdown proteins implicated in the CNS of Huntington's disease (HD) and Tau proteins in mouse models (DeVos et al. (2013) JNeurosci 33 : 12887-12897; Yu et al. (2012) Cell 150: 895-908). These RNA sequences will require the same packaging vehicles.

Linear polyethylenimine (LPEI) has been demonstrated to be a versatile carrier for gene therapy applications (Bonnet et al. (2008) Pharm Res 25: 2972-2982; Jager et al. (2012) Chem Soc Rev 41 : 4755-4767). Studies have highlighted the ability of LPEI-based carriers to complex with DNA and RNA, forming complexes with an average diameter in tens to a couple of hundreds of nanometers, to deliver targets of interest in vitro and in vivo to modulate gene expression (Jager et al. (2012) Chem Soc Rev 41 : 4755-4767; H5bel and Aigner (2013) Wiley Interdiscip Rev Nanomed Nanobiotechnol 5: 484-501). Nanoparticles prepared with LPEI and plasmid DNA have successfully mediated gene expression in the mouse CNS (Goula et al. (1998) Gene Ther 5: 712-717). In this study, specific gene knockdown is demonstrated in mouse neuroblastoma N2a cells using LPEI with an average molecular weight (MW) of 17 kDa (LPEI_17k) to encapsulate siRNA targeting BACE1 or APP. To improve biocompatibility of LPEI, polyethylene glycol (PEG, M_n = 10 kDa) was grafted to LPEIi¾ at different grafting densities, and a method was developed to package siRNA into micellar nanoparticles with different shapes including wormlike, rod-like, and spherical. Here, methods for varying the shapes of these nanoparticles by varying the structural parameters of the carriers and assembly condition between the copolymer carrier and siRNA are described, and then the in vivo efficacy and safety of these shaped nanoparticles in terms of targeting BACE1 in the CNS of mice are compared.

Results

To improve the biocompatibility of LPEI/siRNA nanoparticles, PEGio_k was grafted on to LPEIi¾ with varying grafting degrees. In most of the reported work, higher N/P ratios (i.e., molar ratio of amines in the LPEI to phosphate groups in the siRNA) have been used to maximize siRNA packaging and transfection (Shim and Kwon (2009) Bioconjug Chem 20: 488^199). siRNA nanoparticles were prepared with LPEIH at a relatively lower N/P ratio of 5, so as to limit cytotoxic effects in both in vitro and in vivo contexts (Zheng et al. (2012) ACS Nano 6: 9447-9454). Under these conditions, siRNA can be effectively condensed. Using transmission electron microscopy (TEM), it was observed that nanoparticles made with LPEI-g- PEG at increasing PEG grafting density of 0.6, 0.8, or 1.2% displayed a propensity to form respectively wormlike, rod-like, or spherical shaped nanoparticles (FIG. 7A, FIG. 7B, and FIG. 7C). To prevent relaxation of the complexes and release of siRNA from micelles in physiological media prematurely, and improve delivery efficiency of their payload to the cytoplasm of target cells, a disulfide crosslinking strategy was adopted (Jiang et al. (2010) Adv Mater 22: 2556-2560; Lee et al. (2009) Nano Lett 9: 2402-2406). These stabilized micellar nanoparticles showed high colloidal stability in 150 mmol/1 NaCl solution, with no appreciable increase in size after incubation for 4 hours at room temperature (FIG. 7D). In contrast, LPEI_17k/siRNA particles exhibited a slightly larger size than the LPEI-g-PEG/siRNA micelles, but were prone to significant aggregation in 150 mmol/1 of NaCl (FIG. 7D). Similarly, the crosslinked LPEIi_7K/siRNA nanoparticles also showed severe aggregation in NaCl solution (FIG. 8A and FIG. 8B). The findings suggest that the condensation facilitated by the PEG grafts on the LPEI backbone is essential for the stabilization of siRNA-loaded nanoparticles, an important factor that may impact on in vivo delivery of the siRNA payload.

It is interesting to note that the shape of the nanoparticles is also dependent on the ratio of copolymer to siRNA in the preparation, effectively measured by the N/P ratio. Using LPEIi₇k-g-0.6%-PEGiok as an example, it was observed that minor deviations in N/P ratio from 3 to 6 significantly influenced the shape of complex nanoparticles. TEM imaging for all particles that were stabilized with disulfide crosslinking confirmed that as the N/P ratio increased from 3 to 6, the nanoparticles transitioned from a worm-like morphology at N/P ratio of 3 to a rod-like morphology at N/P ratio of 6 (FIG. 9A, FIG. 9B, FIG. 9C, and FIG. 9D). Both the PEG grafting degree and ratio of LPEI to siRNA can be varied to effectively control the shape of siRNA-loaded micellar nanoparticles.

Before assessing the effect of nanoparticle shape on delivery of siRNA to knockdown targets of interest in the CNS, the appropriate siRNA sequences were screened for and the ability of the siRNA-loaded nanoparticles to knockdown BACE1 and APP in a cell culture model, namely, N2a cells which endogenously express both genes, was evaluated (Li et al. (2006) FASEB J20: 285-292). It was first verified that LPEIn_k complexed with siRNA against either BACE1 or APP, formed nanoparticles with an average diameter of below 100 nm (FIG. 10A, FIG. 10B, FIG. IOC, FIG. 10D, FIG. 10E). These nanoparticles showed successful delivery of siRNA to the cytoplasm in N2a cells (FIG. 1 ID). After screening multiple siRNAs targeting either BACE1 or APP by transfection of N2a cells (data not shown), two top candidates were selected, BACE33 and APP35, for further evaluation. Protein blot analysis revealed that while naked siRNAs were unable to alter the protein levels of their targets, LPEIn_k/siRNA complexes encapsulating BACE33 or APP35 reduced the level of B ACE 1 or APP, respectively, by 63.3 ± 25.4% or 75.6 ± 10.2% of nontransfected control (FIG. 11A, FIG. 1 IB, and FIG. 1 1C). It was noted that the knockdown oiBACEl by BACE33 did not alter the level of APP, and neither did the knockdown οΐΑΡΡ by APP35 affect the level oiBACEl. Moreover, transfections performed with LPEIi_7K/siRNA nanoparticles at a dose of 5 μg of siRNA did not elicit cytotoxicity as judged by the viability of transfected cells (FIG. 1 IE).

Empirically, it was determined that an optimal dose for knockdown of BACE1 is between 3 to 5 μg of siRNA (FIG. 11F). Next, the effect of nanoparticle shape on the delivery of siRNAs to cells of the CNS was assessed globally. Intraventricular infusions of LPEI siRNA nanoparticles and shaped micellar nanoparticles were performed in awake and freely moving mice (FIG. 12A), a delivery approach thought capable of achieving global distribution of payload in the brain (Yu et al. (2012) Cell 150: 895-908; Wang et al. (2008) J Biol Chem 283: 15845-15852). Mice were initially infused with up to 64 μg of siRNA in the right lateral ventricle (referred as the ipsilateral side) over a 7-day period (FIG. 12B). Unfortunately, such effort of using optimized formulations from the in vitro studies did not lead to reduction in levels of BACE1 (FIG. 13 A, FIG. 13B, and FIG. 13C). Initial in vivo pilot studies were performed with LPEI/siRNA nanoparticles at N/P ratios of 10 and 20, with the reasoning that the knockdown efficiency would be higher for higher N/P ratios. Consistent with other studies (Williford et al. (2014) Annu Rev Biomed Eng 16: 347-370), significant toxicity was noticed leading to a high fatality rate in the experimental group, where nanoparticles with an N/P ratio of 20 exhibited a higher level of toxicity than that with an N/P ratio of 10. Therefore, a low N/P ratio of 5 was used so as to minimize the amount of LPEI in nanoparticles to mitigate toxicity effects in the CNS. The focus was on evaluating the efficiency of LPEIi_7K-g-PEGio_k/siRNA micellar nanoparticles stabilized with disulfide

crosslinking, with the hypothesis that PEG corona on micellar nanoparticles could reduce toxicity while providing a better opportunity for siRNA delivery due to higher colloidal and complex stability. For all of the LPEIn_k-g-PEGio_k/siRNA nanoparticles in N2a cells, the viability was greater than 95% when compared with the

untransfected control (FIG. 1 IE). However, these LPEIn_k-g-PEGio_k/siRNA micellar nanoparticles showed minimal transfection and knockdown efficiency in vitro. Given that the in vitro results do not typically match the in vivo transfection efficiency for PEGylated carriers (H5bel and Aigner (2013) Wiley Inter discip Rev Nanomed Nanobiotechnol 5: 484-501 ; Mishra et al. (2004) Eur J Cell Biol 83 : 97-1 11), the in vivo delivery efficiency of these micellar nanoparticles with spherical, rod-like and worm-like shapes to the CNS continued to be assessed.

To determine whether nanoparticles can be transported into the brain parenchyma, the fate of fluorescently labeled siRNA encapsulated in nanoparticles was followed in the brain of mice, following an intraventricular infusion for two days. To facilitate identification of neurons in the brain, mice genetically encoded with GFP in which neurons are sparsely marked by GFP in the cytoplasm were used (Feng et al. (2000) Neuron 28: 41-51). Proximal to the lateral ventricle, a gradient of fluorescently labeled siRNA was observed emanating from the infusion site toward the brain parenchyma (FIG. 14). The accumulation pattern of the siRNA strongly suggests that these nanoparticles were able to gain access to the cytoplasm of cells in the brain parenchyma.

To assess potential untoward side effects of nanoparticles infused into brains of mice up to one week, glial cells were examined, which are normally activated and migrate to the site of injury in the brain (Holguin et al. (2007) JNeurosci Methods 161 : 265-272). Since cannulation of the right lateral ventricle would lead to the injury of tissue in the immediate vicinity, it was first confirmed that similar activation of glial cells at the injury site occurred for animals infused with vehicle (5% glucose solution) or those with nanoparticles (FIG. 15A, FIG. 15B, FIG. 15C, FIG. 15D, FIG. 15E, and FIG. 15F). It was noted that in regions away from the site of injury, a decrease in glial cell activation was observed (FIG. 15A, FIG. 15B, FIG. 15C, FIG. 15D, FIG. 15E, and FIG. 15F). No significant difference in glial cell activation was seen in the contralateral side of brains between both sets of mice (data not shown). While these results confirmed that LPEIn_k-g-PEGio_k/siRNA nanoparticles did not elicit an untoward response over a 2-day period, any potential impact for mice infused for at least 7 days was also examined. Using markers of astrocytic and microglial activation, a difference in activation of both cell types in the ipsilateral hippocampus of mice infused with rod shaped nanoparticles as compared to control was not observed (FIG. 12C, FIG. 12D, FIG. 12E, and FIG. 12F). Likewise, no difference was observed for other types of shaped nanoparticles either (FIG. 16A, FIG. 16B, FIG. 16C, FIG. 16D, FIG. 16E, FIG. 16F, FIG. 16G, FIG. 16H, FIG. 161, FIG. 16J, FIG. 16K, FIG. 16L, FIG. 16M, and FIG. 16N). Taken together with the observation that microglial and astrocytic cells continued to maintain their normal morphology, these findings suggest that these micellar nanoparticles do not induce significant inflammation and cytotoxicity in the brain when infused for over 1 week.

Interestingly, while worm-like and spherical nanoparticles did not alter BACEl levels, infusion of rod-shaped nanoparticles into the lateral ventricle led to the knockdown of BACEl by 36.8 ± 4.8% and 42.8 ± 2.9% within the ipsilateral and contralateral side of the cortex, respectively, as compared to the vehicle infused control (FIG. 17A, FIG. 17C, and FIG. 17D). In the ipsilateral hippocampus, spherical, rod-like, and worm-like nanoparticles reduced BACEl levels by 24.1 ± 4.1%, 38.2 ± 3.4%, and 33.3 ± 6.7%, respectively (FIG. 17B, FIG. 17E, and FIG. 17F). In the contralateral hippocampus, the corresponding BACEl knockdown efficiencies were 28.9 ± 4.1%, 35.6 ± 5.9%, and 24.2 ± 4.5%, respectively, for spherical, rod-like, and worm-like nanoparticles. Together, these findings suggest that the shape of nanoparticles appears to influence their efficiency for delivery to cells in the CNS, and the rod-like particles are most efficient among this series. To further evaluate how extensive these nanoparticles could distribute throughout the CNS, their ability to knockdown BACEl in the brainstem and spinal cord, regions that are farther away from the site of infusion, was examined. It too was observed that the nanoparticles were able to significantly reduce levels of BACEl in the brainstem and spinal cord of mice (FIG. 18A, FIG. 18B, FIG. 18C, FIG. 18D, FIG. 18E, and FIG. 18F). These findings thus establish that efficient siRNA delivery to the CNS can be achieved by shaped micellar nanoparticles based on the LPEI_17k-g-PEGio_k/siRNA nanoparticle platform.

Discussion

Despite the enormous therapeutic potential of siRNAs for treating

neurological diseases, delivery of these molecules, either through local or systemic administration, remains a great challenge (Kanasty et al. (2013) Nat Mater 12: 967- 977). Tremendous progress has been made towards developing suitable carriers for siRNA as therapeutics which in the form of nanoparticles have reached clinical trials (Coelho et al. (2013) N Engl J Med 369: 819-829). Besides particle surface properties, and charge density, nanoparticle shape has been shown to be a key parameter towards improving the delivery efficiency (Venkataraman et al. (201 1) Adv Drug Deliv Rev 63: 1228-1246). Studies have shown how different shapes utilize differing mechanisms to gain entry into cells (Gratton et al. (2008)

Proc Natl Acad Sci USA 105: 1 1613-11618; Agarwal et al. (2013) Proc Natl Acad Sci USA 110: 17247-17252). While exciting progress has been made in terms of manipulating RNA sequences into a variety of geometries, shape control of the RNA species encapsulated within nanoparticles has been inaccessible (Shopsowitz et al. (2014) Small 10: 1623-1633; Afonin et al. (2014) Acc Chem Res 47: 1731-1741; Afonin et al. (2012) Nano Lett 12: 5192-5195). A recent study showed that rod shaped particles accumulated to a greater extent in the lung and brain vasculature when infused intravenously as compared to spherically shaped particles (Kolhar et al. (2013) Proc Natl Acad Sci USA 1 10: 10753-10758). Recent work showing that the shape of DNA-encapsulated nanoparticles is an important factor to influence transfection efficiency in a rat liver model supports the notion that the shape of nanoparticles could be an important determinant for their payload delivery efficiency (Jiang et al. (2013) Adv Mater 25: 227-232). Pioneering work involving the use of DNA as building blocks have been extended to RNA and more recently to long R Ai (microsponges) that were developed in an effort to overcome challenges of packaging short pieces of siR A (Shopsowitz et al. (2014) Small 10: 1623-1633; Lee et al. (2012) Nat Nanotechnol 7: 389-393). These RNA species when encapsulated by a polycationic carrier typically form spherical particles. While shape control using RNA as a structural building block has been achieved (Afonin et al. (2012) Nano Lett 12: 5192-5195), the control of nanoparticle shape with RNA as the functional payload has not been demonstrated. The finding on the ability to control siRNA nanoparticle shape using LPEI-g-PEG copolymer carriers establishes the first evidence that the shape of polycation/siRNA micellar

nanoparticles can be varied in a systematic fashion. This study confirmed that increasing the PEG grafting density leads to more condensed spherical morphology, whereas lower grafting density yields rod- and worm-like micelles. This observation can be explained using the traditional micelle packing models for amphiphilic diblock copolymer micelles assembled in aqueous media (Israelachvili, Intermolecular and Surface Forces. 3rd edition. Academic Press, Burlington, MA, 201 1). Assuming that the degrees of condensation between the LPEI backbone and siRNA are similar for graft copolymers with different PEG grafting densities under the same N/P ratio, since the grafting densities are relatively low (< 2%), condensation of siRNA with copolymers having a higher PEG grafting density generates micellar nanoparticles with a higher PEG surface density, thus favoring the formation of spherical micelles. This is analogous to micelle assembly, where the shape control is governed by the volume ratio of the hydrophilic (corona) to hydrophobic (core) blocks— higher ratio favors spherical micelle formation and lower ratio yields worm-like micelles

(Israelachvili, Intermolecular and Surface Forces. 3rd edition. Academic Press, Burlington, MA, 201 1). The trend observed by varying N/P ratio is also consistent with this interpretation: a higher N/P ratio leads to higher PEG density on micelle surface, favoring spherical micelle formation, although it is surprising that such a small range of N/P ratio variation from 3 to 6 can lead to significant shape variation. Using this method, nanoparticles were able to be generated with relatively low N/P ratios compared with the higher amount of transfection agents used in nanoparticle formulations (Shim and Kwon (2009) Bioconjug Chem 20: 488-499). This is advantageous in terms of better biocompatibility and lower cytotoxicity of the nanoparticles prepared for transfection applications (Zheng et al. (2012)

ACS Nano 6: 9447-9454). The overall strategy also relies on stabilization of the shaped nanoparticles using a reversible disulfide crosslinking scheme that has been widely adopted by other DNA and siRNA delivery platforms (Jiang et al. (2010) Adv Mater 22: 2556-2560). This method ensures that the prepared nanoparticles will maintain their size and shape in serum containing medium, important to in vivo administration. The availability of a panel of shape-controlled nanoparticles will enable future studies to explore shape-dependent transport in vivo. This method relying on the control of copolymer structure and concentrations of the copolymer and siRNA solutions is thus straightforward to scale up for production.

Previous studies have highlighted challenges involved in designing a suitable method to target the key enzymes involved in the APP pathway (Menting and Claassen (2014) Front Aging Neurosci 6: 165; Reiman (2014) Lancet Neurol 13 : 3- 5). Since mechanism-based toxicities are associated with inhibition of γ-secretase (Li et al. (2007) J Neurosci 27: 10849-10859), the focus was on developing a suitable delivery vehicle for siRNA targeting BACE1 and APP. Previous studies employed RNAi strategies delivered by viral vectors to reduce levels oiBACEl, which emerged as a powerful tool to deliver short hairpin RNA to decrease protein levels (Singer et al. (2005) Nat Neurosci 8: 1343-1349; Laird et al. (2005) J Neurosci 25: 11693- 11709). The nonviral siRNA delivery approaches can potentially mitigate putative safety concerns associated with viral vectors, such as insertional mutagenesis and the risk of inflammation (Kamat et al. (2013) Mol Cancer Ther 12: 405-415). One recent report showed a nanoparticle delivery system using exosomes, with rabies virus glycoprotein (RVG) as a targeting ligand, can reduce the level oiBACEl when delivered to the brain (Alvarez-Erviti et al. (2011) Nat Biotechnol 29:341-345). The study here confirms that the series of shape-controlled micellar nanoparticles are nontoxic to brain tissue as judged by the lack of astrocytic or glial cell activation in response to nanoparticle infusion over a 7-day period. After validating the ability of nanoparticles to deliver siRNA to modify levels oiBACEl and APP, two different proteins in the APP processing pathway of AD, in an N2a cell culture model, their efficacy was evaluated in the CNS of mice by targeting BACE1. Since AD is a neurodegenerative disorder that globally affects the brain, the focus was on ensuring that the nanoparticles were distributed throughout the brain as opposed to a local infusion close to the site of interest. In contrast to several studies focused on inducing RNAi in the CNS of mice (Passini et al. (201 1) Sci Transl Med 3 : 72ral 8; Lima et al. (2012) Cell 150: 883-894), this discovery of shaped nanoparticles provides the opportunity to evaluate whether shape of nanoparticles is a major determinant for efficient delivery of siRNA throughout the CNS. This study establishes that rod-liked nanoparticles exhibited higher efficiency in reducing BACE1 levels in the ipsilateral and contralateral side of the brain. Interestingly, the reduction in BACE1 levels observed in the hippocampus was not significantly different among various shaped nanoparticles. This could be attributed to the proximity of the hippocampus to the lateral ventricles, which mimics a local infusion process. Previous work using mouse models of AD has supported the notion that moderate reductions in BACE1 would prevent potential mechanism-based toxicity while providing beneficial effects in the brain (Chow et al. (2010) Sci Transl Med 2: 13ral). The modest reduction oiBACEl afforded by the presently disclosed nanoparticle delivery system in the CNS (35- 45%) would be amenable for design of a safe and effective therapeutic strategy to target BACE1 for AD. Moreover, the findings of knockdown in the brain stem and spinal cord, regions that are farther away from the infusion site, further support the notion that these shaped nanoparticles achieved broad distribution and delivery of siRNAs in the CNS.

Although it has been shown here that rod-shaped particles work best in this animal model following intraventricular delivery, it remains to be demonstrated as to whether the wormlike and spherical nanoparticles, at varying N/P ratios, would be more suitable for other tissue targets or when administered through a different delivery route, such as intrathecal infusion or intravenous delivery. Worm-like micellar nanoparticles with longer circulation dynamics in rodent models have been used for intravenous delivery (Jiang et al. (2013) Adv Mater 25: 227-232; Geng et al. (2007) Nat Nanotechnol 2: 249-255; Osada et al. (2012) Biomaterials 33 : 325-332). Decorating nanoparticles with targeting ligands of the insulin or transferrin receptor or cell penetrating peptides have been established as promising approaches to improve delivery of cargo to cells of interest (Kamide et al. (2010) Int J Mol Med 25: 41-51 ; Atwal et al. (201 1) Sci Transl Med 3: 84ra43). These approaches also may be coupled with shape-controlled siRNA nanoparticles. As siRNA and antisense therapeutic strategies continue to mature and move into clinical trials using highly optimized sequences, these shape-controlled nanoparticles and their unique properties may provide new opportunities to optimize RNA therapeutic delivery for a variety of disease targets.

In conclusion, it has been shown here that micellar nanoparticles with worm- and rod-like, and spherical shapes can be prepared by self-assembly of the complexes between siRNA and LPEI-g-PEG copolymer carriers. The PEG corona and reversibly crosslinked core of the micelles enable these nanoparticles to be stable under physiological conditions. Interestingly, these micellar nanoparticles revealed differences in knockdown capability following infusion into the lateral ventricles in mice with the rod-like micelles showing the most effective and selective knockdown of a key therapeutic target in AD. siRNA delivery strategies leveraging shape as a tunable parameter creates a translatable platform for RNAi therapeutics.

REFERENCES

All publications, patent applications, patents, and other references mentioned in the specification are indicative of the level of those skilled in the art to which the presently disclosed subject matter pertains. All publications, patent applications, patents, and other references are herein incorporated by reference to the same extent as if each individual publication, patent application, patent, and other reference was specifically and individually indicated to be incorporated by reference. It will be understood that, although a number of patent applications, patents, and other references are referred to herein, such reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

Alvarez-Erviti, L.; Seow, Y.; Yin, FL; Betts, C; Lakhal, S.; Wood, M.J., Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nat. Biotechnol. 2011, 29: 341-345.

Jensen, S.A.; Day, E.S.; Ko C.H.; Hurley, L.A.; Luciano, J.P.; Kouri, F.M., Spherical nucleic acid nanoparticle conjugates as an RNAi-based therapy for glioblastoma. Set Transl. Med. 2013, 5:209ra 152. Jiang, X.; Qu, W.; Pan, D.; Ren, Y.; Williford, J.M.; Cui, H.; Luijten, E.; Mao, H.Q., Plasmid-templated shape control of condensed DNA-block copolymer nanoparticles. Adv. Mater. 2013. 25(2): 227-232.

Lee, H.; Lytton-Jean, A.K.R.; Chen, Y.; Love, K.T.; Park, A.I.; Karagiannis, E. D., Molecular self-assembled nucleic acid nanoparticles for targeted in vivo siR A delivery, Nat. Nanotechnol. 2012, 7(6): 389-393.

Although the foregoing subject matter has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be understood by those skilled in the art that certain changes and modifications can be practiced within the scope of the appended claims.

Claims

THAT WHICH IS CLAIMED:

1. A method for preparing a shape-controlled nucleic acid nanoparticle, the method comprising:

providing a copolymer solution comprising a block copolymer or a graft copolymer of a water soluble, non-charged polymer and a polycation in a first solvent, wherein the water soluble, non-charged polymer and the polycation each have a structure and a molecular weight; and

mixing the copolymer solution with a solution of nucleic acid at a

predetermined nucleic acid to copolymer ratio and pH in a second solvent to form a shape-controlled nucleic acid nanoparticle, wherein the nucleic acid has a

predetermined number of base pairs and the first and second solvent can be the same or different; and

wherein the shape-controlled nucleic acid nanoparticle has a shape controlled by one or more of the: structure of the water soluble, non-charged polymer; molecular weight of the water soluble, non-charged polymer; structure of the polycation;

molecular weight of the polycation; number of base pairs of the nucleic acid; ratio of nucleic acid to copolymer; pH; first solvent comprising the copolymer solution and/or the second solvent comprising the nucleic acid solution, and, if the copolymer is a graft copolymer, a graft density thereof.

2. The method of claim 1, wherein the water soluble, non-charged polymer comprises polyethylene glycol (PEG).

3. The method of claim 2, wherein the PEG has a molecular weight ranging from about 500 Da to about 20 kDa.

4. The method of claim 3, wherein the PEG has a molecular weight of about 10 kDa.

5. The method of claim 2, wherein the copolymer is a graft copolymer and the PEG has a graft density ranging from about 0.1% to about 20%.

6. The method of claim 5, wherein the PEG has a graft density selected from the group consisting of a 2% graft density, a 4% graft density, and an 8% graft density.

7. The method of claim 6, wherein the 2% graft density of the PEG results in a worm-shaped nucleic acid nanoparticle, the 4% graft density of the PEG results in a rod-shaped nucleic acid nanoparticle, and the 8% graft density of PEG results in a spherically-shaped nucleic acid nanoparticle.

8. The method of claim 1, wherein the polycation is selected from the group consisting of linear polyethylenimine (LP EI), poly-lysine, poly-arginine, poly- histidine, chitosan, branched PEI, a poly (beta-aminoester), a polyphosphoester, polyphosphoramidate (PPA), and PEG-£-polyphosphoramidate (PEG-PPA).

9. The method of claim 8, wherein the polycation is LPEI.

10. The method of claim 9, wherein the LPEI has a molecular weight ranging from about 2 kDa to about 50 kDa.

11. The method of claim 10, wherein the LPEI has a molecular weight of about 17 kDa.

12. The method of claim 1, wherein the nucleic acid is selected from at least one member of the group consisting of an a guide RNA, siRNA, miRNA, shRNA, antisense RNA, antisense oligonucleotide, ribozyme, CRISPR RNA, and an aptamer.

13. The method of claim 12, wherein the nucleic acid comprises siRNA.

14. The method of claim 13, wherein the siRNA comprises about 25 base pairs.

15. The method of claim 13, wherein the nucleic acid comprises an siRNA sequence that is at least 92% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.

16. The method of claim 1, wherein the nucleic acid decreases the expression of B-site APP cleaving enzyme 1 (BACEl) and/or amyloid-B precursor protein (APP).

17. The method of claim 1, wherein the ratio of nucleic acid to copolymer is measured as copolymer nitrogen to nucleic acid phosphate (N/P ratio) and has a range from about 0.1 to about 20.

18. The method of claim 17, wherein the N/P ratio is less than about 10.

19. The method of claim 1, wherein the pH has a range from about 1 to about 7.5.

20. The method of claim 1, wherein the first and/or second solvent is water or in a mixture comprising water and a water-miscible solvent selected from the group consisting of dimethylformamide (DMF), dimethyl sulfoxide (DMSO), dioxane, and tetrahydrofuran (THF).

21. The method of claim 20, wherein the water-miscible solvent is 70%

DMF.

22. The method of claim 21, further comprising removing the water- miscible solvent from the nucleic acid nanoparticle.

23. The method of claim 22, further comprising crosslinking the nucleic acid nanoparticle.

24. The method of claim 1, wherein the shape of the nucleic acid nanoparticle is selected from the group consisting of worm-shaped, spherically- shaped, and rod-shaped.

25. A nucleic acid nanoparticle prepared by the method of claim 1.

26. The nucleic acid nanoparticle of claim 25, wherein the nucleic acid is selected from at least one member of the group consisting of a guide RNA, siRNA, miRNA, shRNA, antisense RNA, antisense oligonucleotide, ribozyme, CRISPR RNA, and an aptamer.

27. The nucleic acid nanoparticle of claim 25, wherein the nucleic acid comprises siRNA.

28. A method for treating a disease or condition, the method comprising administering to a subject in need of treatment thereof, a shape-controlled nucleic acid nanoparticle of claim 1 , or a pharmaceutical composition thereof, in an amount effective for treating the disease or condition.

29. The method of claim 28, wherein the nucleic acid is selected from at least one member of the group consisting of a guide RNA, siRNA, miRNA, shRNA, antisense RNA, antisense oligonucleotide, ribozyme, CRISPR RNA, and an aptamer.

30. The method of claim 29, wherein the nucleic acid comprises siRNA.

31. The method of claim 30, wherein the nucleic acid comprises an siRNA sequence that is at least 92% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.

32. The method of claim 28, comprising administering the shape- controlled nucleic acid nanoparticle to the brain and/or spinal cord of the subject.

33. The method of claim 28, wherein the disease or condition comprises a neurodegenerative disease.

34. The method of claim 33, wherein the neurodegenerative disease is selected from the group consisting of Amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Parkinson's disease, Alzheimer's disease,

Huntington's disease, and dementia with Lewy Bodies.

35. The method of claim 28, further comprising the knockdown of one or more genes.

36. The method of claim 28, wherein the nucleic acid decreases the expression of β-site APP cleaving enzyme 1 (BACEl) and/or amyloid-β precursor protein (APP).