WO2011087804A2 - Peptide-polynucleotide compositions, and methods for transfecting a cell with dna and treatment of neurodegenerative disease - Google Patents

Abstract

Peptide-polynucleotide compositions and conjugates for the treatment of neurodegenerative disease are provided. The compositions and conjugates contain a peptide having one or more of a rabies virus glycoprotein domain, a nuclear localization signal domain, and a DNA condensing domain that is linked either covalently or electrostatically to polynucleotides such as shRNA. Methods of transfecting a cell with DNA, methods of increasing shRNA expression in target cells of a mammal, and methods of treatment are also provided, which include delivery of the peptide-polynucleotide compositions and conjugates of the invention.

Description

PEPTIDE-POLYNUCLEOTIDE COMPOSITIONS, AND METHODS

FOR TRANSFECTING A CELL WITH DNA AND TREATMENT OF NEURODEGENERATIVE DISEASE

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of the effective filing date of U.S.

Provisional Patent Application No. 61/288,793, filed on December 21, 2009, the entire contents of which are hereby incorporated by reference.

REFERENCE TO SEQUENCE LISTING [0002] This application contains a Sequence Listing submitted as an electronic .txt file. The information contained in the Sequence Listing is hereby incorporated by reference.

BACKGROUND OF THE INVENTION [0003] Nucleotides conjugated to cell penetrating peptides can be transfected into cells for gene therapy. Transfection is the process of introducing nucleic acids into cells by non-viral methods. Generally, a therapy using this approach involves the therapeutic regulation of gene expression by using RNA interference (RNAi), which can mediate sequence-selective suppression of gene expression in a variety of eukaryotes by introducing short RNA duplexes with sequence homologies to the target gene. Small, interfering RNA (siRNA) coding a specific gene is typically used in RNAi treatment. siRNA is a class of double-stranded RNA molecules that can, for example, interfere with the expression of a specific gene. Use of siRNA inhibits a gene's expression, where the gene is responsible for advancement of a disease or infection.

[0004] It has been shown that siRNA molecules administered intravenously can be delivered to the brain using a peptide sequence consisting of the glycoprotein domain derived from the rabies virus. Kumar et al. (Transvascular delivery of small interfering RNA to the central nervous system, NATURE, Jul. 2007; 448(7149): 39-43, e-pub June 17, 2007). Delivery of siRNA to the brain using the vascular system has the potential advantage of widespread distribution of the siRNA to all anatomical regions of the brain. However, delivery of siRNA via the rabies virus glycoprotein for purposes of a therapy based on RNA interference may have limitations. First, the effect of siRNA in suppressing a target gene in cells is limited to a period of time after the siRNA has been delivered; once the siRNA has been degraded in the cell, the suppression of the target gene does not persist. Therefore, use of siRNA to treat a patient for a chronic disease may require frequent or continuous administration of the siRNA composition to the patient. Second, the rabies glycoprotein is an amino acid sequence foreign to the human body. Therefore, frequent or continuous delivery of a peptide sequence consisting of this glycoprotein systemically would likely cause an immune reaction in the patient, which may compromise the effectiveness of the therapy and may have other undesirable adverse effects. Therefore, when a treatment needs to be persistent in the brain of a mammal, desirably, the therapy needs to be delivered only once or infrequently at intervals between treatments being months to years or the delivery method must allow for continuous or chronic infusion at a site in the mammal where an immune response is less likely to affect the effectiveness or the therapy or have adverse effects.

[0005] One approach of gene delivery using the 29-amino-acid peptide derived from the rabies virus glycoprotein (RVG29) conjugates the peptide to polyamidoamine dendrimers (PAMAM) using a bifunctional polyethylene glycol (PEG) molecule. Liu Y., et al., Brain- targeting gene delivery and cellular internalization mechanisms for modified rabies virus glycoprotein RVG29 nanop articles, BIOMATE IALS, Sep. 30, 2009; 30(25): 4195-4202, e-pub May 20, 2009. The resulting PAMAM-PEG-RVG29 molecules are then associated in a non- covalent way ("complexed") with DNA, creating PAMAM-PEG-RVG29/DNA complexes for delivery of the DNA to the cells of a mammal by administration into the vasculature of the mammal. A limitation to this approach is that the peptide-DNA complexes can become trapped in the lysosomes or other vesicles of the cell following endocytosis, such that the DNA fails to pass into the nucleus of the cell, where it can be utilized {see Liu et al., Figure 5). Another limitation is that the molecular domains responsible for delivery of the DNA into cells and cell nuclei are not covalently attached to the DNA. As a result, the delivery of the DNA may be inefficient or subject to unpredictable and uncontrollable variability from administration to administration.

[0006] Hence, there is a need for a more effective treatment approach that provides longer lasting gene suppression per administration, so that the composition may be administered once or, at most, infrequently. There is also a need for covalently conjugating a polynucleotide to a delivery peptide for delivery of some gene therapies. Peptides and peptide-polynucleotide compositions and methods using those compositions for the delivery of DNA encoding for short, hairpin RNA (shRNA) and for the treatment of neurological diseases are desired. There is also a need to provide a means to deliver a single or infrequent dose of an RNA-interference-based therapy. The need extends to delivery of molecules of DNA encoding for short, hairpin RNA (shRNA) with a similar effect as the comparable siRNA in terms of target gene suppression. Treatment options should provide for DNA persisting longer in cells than does externally-delivered siRNA. There is also a need for peptide sequences and peptide-polynucleotide compositions to efficiently associate with DNA and transport DNA into the cell nucleus. In addition, there is a need for peptides and peptide-polynucleotide compositions incorporating a peptide signal that has the function of transporting the DNA into the cell nucleus once the DNA is inside the cell.

SUMMARY OF THE INVENTION

[0007] The invention includes peptide-polynucleotide compositions useful for treatment of neurological diseases. The peptide-polynucleotide compositions of the invention may be in the form of G-x-P-Y or P-x-G-Y, which are peptide-polynucleotide conjugates containing the GP or PG peptides of the invention. G and P are as defined below, x is an optional linker consisting of 0 to 100 glycines and/or polyethylene glycol (PEG), and Y is a polynucleotide consisting of single- or double-stranded DNA or single- or double-stranded RNA. The P domain is preferably selected from the peptides in SEQ ID Nos. 2 and 31. The peptide-polynucleotide composition according to this embodiment is any one of SEQ ID Nos. 3-4, 12, and 28-29.

[0008] Alternatively, the peptide-polynucleotide compositions of the invention may be in the form of G-x-N-Y or N-x-G-Y conjugates, which are peptide-polynucleotide conjugates containing the GN or NG peptide of the invention. G and N are as defined below, x is an optional linker consisting of 0 to 100 glycines and/or polyethylene glycol (PEG), and Y is a polynucleotide consisting of single- or double-stranded DNA or single- or double- stranded RNA. The N domain is preferably selected from the peptides in SEQ ID Nos. 5 and 64-65. The peptide-polynucleotide according to this embodiment is any one of SEQ ID Nos. 9-10 and 15-18.

[0009] Alternatively, the peptide-polynucleotide compositions of the invention may be in the form of G-xi-P-x₂-N-Y, P-xi-N-x₂-G-Y, G-xi-N-x₂-P-Y, P-xi-G-x₂-N-Y, N-xi-G- x₂-P-Y, and N-x₁-P-x₂-G-Y, which are peptide-polynucleotide compositions containing the GPN, PNG, GNP, PGN, NGP, and NPG peptides of the invention. G, P, and N are as defined below, Xi and x₂ are optional linkers consisting of 0 to 100 glycines and/or PEG, and Y is a polynucleotide consisting of single- or double-stranded DNA or single- or double-stranded RNA. The P domain is preferably selected from the peptides in SEQ ID Nos. 2 and 31. The N domain is preferably selected from the peptides in SEQ ID Nos. 5 and 64-65. The peptide- polynucleotide composition according to this embodiment is any one of SEQ ID Nos. 6-8, 14, 20-27, and 39-63. [0010] Optionally, any of the peptides of the invention may be conjugated to one 5' end of the Y polynucleotide, where Y is double-stranded DNA or double- stranded RNA, and a second same peptide of the invention may be conjugated to the 5' end of a complementary strand of Y.

[0011] The polynucleotide Y in the foregoing embodiments of the peptide- polynucleotide compositions of the invention may consist of a Silencer Expression Cassette, such as the SECs of the invention, including the SEC in SEQ ID Nos. 11 and 30. The polyethylene glycol (PEG) molecule in the optional linker preferably has a molecular weight ranging from 2,000 Da to 10,000 Da. The peptide-polynucleotide conjugates may contain at least one amino acid or nucleotide having azide or alkyne functionality. Azide functionality can be obtained by incorporation of a PEG having a terminal azide group wherein the number of PEG repeat groups is from 1 to 150. Alkyne functionality can be obtained from inclusion of a phosphoramidite. The polynucleotide Y may be covalently linked to the peptides of the invention according to the formulas of the different embodiments of the invention. Alternatively, the polynucleotide Y may consist of plasmid DNA that associates with the peptides of the invention.

[0012] Optionally, any of the peptide-polynucleotide compositions of the invention may be further combined with histones that condense the DNA of the peptide-polynucleotide composition. In these embodiments, histones are present in a molar ratio from 1: 1 histones to DNA to 31: 1 histones to DNA, or, generally, from n: l histones to DNA, for each n={x £ M| l < x < 31}. Some embodiments of the ratio of histones to DNA include molar ratios of 6: 1, 15: 1, 30: 1, and 31: 1 histones to DNA.

[0013] The invention also includes methods of making the peptide-polynucleotide conjugates of the invention. The method consists of modifying an amino acid of the peptide to have azide functionality, modifying a nucleotide of the Y polynucleotide to have alkyne functionality, and reacting the modified peptide and the modified polynucleotide to form a 5- membered heterocycle (triazole ring) using a copper catalyst to form a covalent bond between the peptide to the nucleotide. Optionally, the copper catalyst may be stabilized using the stabilizing agent tris-(benzyltriazolylmethyl)amine (TBTA). The amount of TBTA used is preferably in a molar excess of 2: 1 compared to the amount of the copper catalyst. In an embodiment, the amino acid is preferably modified with NHS-(PEO)₄-azide. To impart alkyne functionality, the nucleotide is preferably modified using an alkyne functional phosphoramidite. The alkyne functional phosphoramidite is more preferably 5'-hexynyl phosphoramidite. Azide functionality may alternatively be achieved using any of the following: azidohomoalanine, asidoalanine, 2-amino-5-hexanoic acid, azidophenylalanine, and alkynyl tyrosine.

[0014] The invention also includes methods of transfecting a cell with DNA and methods of increasing shRNA expression in target cells of a mammal through delivery of a polynucleotide of the invention to the mammal. In one embodiment, the peptide- polynucleotide composition contains a nuclear localization signal (NLS) amino acid domain to facilitate delivery of the polynucleotide DNA to the cell nucleus. The compositions of the invention are, in certain embodiments, mixed with histones for effecting increased shRNA expression in target tissue of the mammal. The histones are combined with the polynucleotide DNA in a molar ratio from 1 : 1 histones to DNA to 31: 1 histones to DNA, or, generally, from n: l histones to DNA, for each n={x £ M| l < x < 31}. Some embodiments of the ratio of histones to DNA include molar ratios of 6: 1, 15: 1, 30: 1, and 31: 1 histones to DNA.

[0015] In one embodiment, the peptide-polynucleotide compositions of the invention are administered by injection to the carotid or femoral artery of the mammal or intracranially or intraparenchymally. In one embodiment the compositions of the invention can be delivered by way of a catheter or other delivery device having one end implanted in a vessel or tissue, e.g., the brain by, for example, intracranial or intraparenchymal infusion. For administration to a mammal of the peptide-polynucleotide compositions, the peptide- polynucleotide compositions may additionally be combined with a pharmaceutically acceptable buffer. The pharmaceutically acceptable buffer is preferably an injectable saline solution or a phosphate buffered saline solution. The invention also includes methods of treatment of neurodegenerative disease through administration of any of the peptide- polynucleotide compositions of the invention to a mammal in need thereof. An embodiment of the invention uses a peptide-polynucleotide composition having a G amino acid sequence that facilitates transport of the peptide-polynucleotide composition to tissue of the mammal. Alternatively, naked siRNA is administered to the mammal for treatment of neurodegenerative disease, via the carotid artery. In certain embodiments, the compositions of the invention are mixed with histones for effecting increased shRNA expression in target tissue of the mammal.

[0016] In one embodiment, the peptide-polynucleotide compositions of the invention are administered by way of a catheter or other delivery device implanted in a predetermined tissue site or vessel. The catheter may be operably connected to an infusion pump that pumps the peptide-polynucleotide compositions of the invention into the tissue or vessel and may include a controller for controlling the rate at which the compositions is delivered. In another embodiment, the infusion pump can include a reservoir containing the tracing composition. It is contemplated that the pump can be implantable and the reservoir can be refillable. Systems useful for delivering solutions or compositions to the brain are described in U.S. Patent Application Pub. No. 2005/0048641. The infusion pump can be implantable or may be an external device and can take the form of any pump system, including, but not limited to, a drug reservoir and/or a drug pump of any kind, for example an osmotic pump, an infusion pump, an electromechanical pump, an electroosmotic pump, an effervescent pump, a hydraulic pump, a piezoelectric pump, an elastomeric pump, a vapor pressure pump, or an electrolytic pump. Examples of suitable pumps include the device shown in U.S. Patent No. 4,692,147 (Duggan), assigned to Medtronic, Inc., Minneapolis, Minn., an embodiment of which is commercially available as the Synchromed® infusion pump manufactured by Medtronic, Inc. and Medtronic' s Synchromed® II infusion pump.

[0017] The infusion pump can be implanted below the skin of a patient. Preferably, the pump is implanted in a location where the implantation interferes as little as practicable with activity of the mammal. The pump can be implanted subcutaneously in any medically acceptable area of the human body such as in a subcutaneous pocket located in the chest below the clavicle, in an abdominal subcutaneous pocket, in the mammal's cranium, and the like.

[0018] Methods that use a catheter to deliver a therapeutic agent to the brain generally involve inserting the catheter into the brain and delivering the agent, solution or composition to the desired location. To accurately place the catheter and avoid unintended injury to the brain, surgeons typically use stereotactic apparatus/procedures. (U.S. 4,350,159) During a typical implantation procedure, an incision may be made in the scalp to expose the patient's skull. After forming a burr hole through the skull, the catheter may be inserted into the brain.

[0019] Other delivery devices useful for the methods of this invention include a device providing an access port, which can be implanted subcutaneously on the cranium and through which therapeutic agents may be delivered to the brain, such as the model 8506 ICV Access Port and the 8507 Intraspinal Port, developed by Medtronic, Inc. of Minneapolis, Minn. Two models of catheters that can function with the model 8506 access port include the model 8770 ventricular catheter (Medtronic, Inc.), for delivery to the intracerebral ventricles, which is disclosed in U.S. Patent No. 6,093,180, and the infusion catheter developed by Medtronic, Inc., for delivery to the brain tissue itself (i.e., intraparenchymal delivery), which is described in U.S. Patent Application Publication Nos. 2009/540,444 and 2009/625,751, the teachings of which are incorporated herein by reference. The latter catheter has multiple outlets on its distal end to deliver the therapeutic agent to multiple sites along the catheter path.

[0020] Examples of external pump type delivery devices are described in U.S. patent application Ser. No. 11/211,095, filed Aug. 23, 2005, titled "Infusion Device And Method With Disposable Portion" and Published PCT Application No. WO 01/70307 (PCT/USOl/09139), titled "Exchangeable Electronic Cards For Infusion Devices" (each of which is owned by the assignee of the present invention), Published PCT Application No. WO 04/030716 (PCT/US2003/028769), titled "Components And Methods For Patient Infusion Device," Published PCT Application No. WO 04/030717 (PCT/US2003/029019), titled "Dispenser Components And Methods For Infusion Device," U.S. Patent Application Publication No. 2005/0065760, titled "Method For Advising Patients Concerning Doses Of Insulin," and U.S. Patent No. 6,589,229 titled "Wearable Self-Contained Drug Infusion Device," each of which is incorporated herein by reference in its entirety. The present invention contemplates the aforementioned pumps adapted for use in delivering the compositions of the invention.

[0021] The methods of treatment of the invention preferably use peptide- polynucleotide compositions of the invention where the Y polynucleotide codes for shRNA. Optionally, the shRNA targets any of the following proteins: beta-amyloid cleaving enzyme type 1, gamma-secretase, amyloid precursor protein, a-synuclein, Huntingtin Protein, ataxin- 1, ataxin-2, ataxin-3, atrophin-1, caspase-3, and caspase-9. Alternatively, the shRNA targets any of the following genes: BACE1, PSEN1, APP, SNCA, HD, SCA1, SCA2, SCA3, DRPLA, CASP3, and CASP9. [0022] Embodiments of the method of treatment of the invention include but are not limited to the treatment of each of the following known neurodegenerative diseases: Alzheimer's disease, Parkinson's disease, Huntington's disease, Lewy body dementia, a lysosomal storage disease, or Tay Sachs disease.

[0023] The invention also includes methods of suppressing gene expression in target cells of a mammal. In the method, the cells are transfected with any of the peptide- polynucleotide compositions of the invention or naked siRNA. A preferred embodiment includes a method of suppressing BACE1 expression in neuronal cells of a mammal, where the transfected polynucleotide targets BACE1. An embodiment of the invention uses a peptide-polynucleotide conjugate having a G amino acid sequence that facilitates transport of the peptide-polynucleotide conjugate. In certain embodiments, the compositions used in the method are mixed with histones, which effect greater suppression of target gene expression. The step of administering includes the composition or conjugate being administered intracranially, intraparenchymally, arterially or intravenously. In one embodiment, the method of administration is by injection or by a catheter in fluid communication with a drug delivery pump. Naked siRNA is most preferably administered via the carotid artery of the mammal.

[0024] The invention also includes methods for treating or delaying the onset of neurological symptoms in a mammal with or at risk of developing a neurodegenerative disease. In the method, any of the peptide-polynucleotide compositions of the invention are intracranially or intraparenchymally administered to a mammal. Alternatively, the methods contemplate treating and/or delaying onset or worsening of a symptom in a mammal with or at risk of neurodegenerative disease wherein the peptide-polynucleotide conjugate uses a nuclear localization signal amino acid sequence (N) to facilitate transport of the polynucleotide into the cell nucleus. In one embodiment, the peptide-polynucleotide composition uses a G amino acid sequence to facilitate transport of the peptide- polynucleotide conjugate.

[0025] The method for treating or delaying the onset of symptoms in a mammal with or at risk of developing a neurodegenerative disease may be used for, in different embodiments, the following neurodegenerative diseases: Alzheimer's disease, Parkinson's disease, Huntington's disease, Lewy body dementia, a lysosomal storage disease, or Tay Sachs disease. These methods preferably use peptide-polynucleotide compositions of the invention where the Y polynucleotide codes for shRNA. Optionally, the shRNA targets any of the following proteins: beta-amyloid cleaving enzyme type 1, gamma- secretase, amyloid precursor protein, a-synuclein, Huntingtin Protein, ataxin-1, ataxin-2, ataxin-3, atrophin-1, caspase-3, and caspase-9. Alternatively, the shRNA targets any of the following genes: BACE1, PSEN1, APP, SNCA, HD, SCA1, SCA2, SCA3, DRPLA, CASP3, and CASP9.

[0026] The invention also includes methods for enhancing siRNA uptake by target cells. In the method, the a peptide-polynucleotide conjugate containing a G domain and a polynucleotide consisting of single- or double-stranded DNA or single- or double-stranded RNA is administered via the carotid or femoral artery of a mammal. Preferably, the polynucleotide is siRNA. Optionally, the DNA is combined with histones in a molar ratio from 1: 1 to 31: 1, as previously described.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is a photograph of an agarose gel in which GP peptides associated with DNA or G peptides associated with DNA were subject to electrophoresis, showing that the DNA is associated with the GP peptides, and thus retained at the upper end of the gel, at a lower ratio of GP peptides to DNA than the ratio needed for G peptides to DNA. [0028] Figure 2 is a photograph of an agarose gel in which GP peptides associated with siRNA or G peptides associated with siRNA were subject to electrophoresis, showing that the siRNA is associated with the GP peptides and the G peptides about equivalently.

[0029] Figure 3 is a photograph of an agarose gel in which GPN peptides associated with plasmid DNA, GP peptides associated with plasmid DNA, or G peptides associated with plasmid DNA were subject to electrophoresis. Figure 3 shows that the GPN peptide associates with plasmid DNA sufficiently to inhibit DNA migration into the gel and that it does so at a lower ratio of peptide to DNA than the other peptides.

[0030] Figure 4 is a collection of photographs of Neuro2a cells or 293T cells that have been transfected in vitro with a plasmid encoding for green fluorescent protein using G peptides or the GP or GPN peptides of the present invention.

[0031] Figure 5 is a graph showing suppression of BACE1 mRNA expression in

Neuro2a cells transfected with DNA comprising a Silencer Expression Cassette encoding for a shRNA targeting BACE1, using GPN peptides of the invention at different ratios of peptide to DNA.

[0032] Figure 6 is a graph showing relative amounts of DNA and transcripts of RNA from DNA comprising a Silencer Expression Cassette encoding for the shRNA in the brain tissue of a mouse after direct intracranial infusion of the DNA with various agents including the peptides of the invention.

[0033] Figure 7 is a collection of photographs showing (1) fluorescently labeled DNA comprising a Silencer Expression Cassette in the brain tissue of a mouse after direct intracranial infusion of the DNA with peptides of the invention and (2) nuclear staining of cells in the same brain tissue and co-localization of the fluorescence with the nuclear staining, which indicates cellular uptake of the DNA delivered by the peptides of the invention. [0034] Figure 8 is a graph showing the level of expression of shRNA transcripts in brain tissue from the hippocampal region of mice brains after infusion of DNA comprising a Silencer Expression Cassette encoding the shRNA into the carotid artery with various peptides of the invention.

[0035] Figure 9 is a collection of graphs showing the output of high-pressure liquid chromatography analysis of the peptide -polynucleotide conjugates of the invention that were formed using "click chemistry."

[0036] Figure 10 is a photograph of an agarose gel in which DNA comprising a

Silencer Expression Cassette and the peptide-polynucleotide conjugates of the invention that were formed using a "click chemistry" reaction have been subjected to electrophoresis.

[0037] Figures 11(a) to 11(g) show the level of expression of shRNA in Neuro2a cells transfected with DNA comprising a Silencer Expression Cassette encoding for the shRNA or with the peptide-polynucleotide conjugates of the invention and indicating enhanced transfection of Neuro2a cells by the peptide-polynucleotide conjugates of the invention.

[0038] Figure 12 is a graph showing the level of expression of shRNA transcripts in brain tissue from the hippocampal region of mice brains after infusion of DNA comprising a Silencer Expression Cassette encoding the shRNA into the carotid artery with various peptides of the invention, including GN-SEC conjugates.

[0039] Figure 13 is a graph showing the level of expression of shRNA transcripts in the left hippocampus of mice brains after delivery into the left carotid artery of the mice of DNA comprising a Silencer Expression Cassette encoding for the shRNA or a peptide- polynucleotide conjugate of the invention.

[0040] Figure 14 illustrates a "click chemistry" reaction between an azide-functional peptide and an alkyne-functional DNA molecule. [0041] Figure 15 depicts the condensing of DNA by histones into a single nucleosome core particle.

[0042] Figure 16 depicts multiple nucleosome core particles condensing a sequence of DNA.

[0043] Figure 17 illustrates the relative expression of SEC 1749 in N2A cells treated with G-N-SEC1749 or SEC1749 with or without histones. It is noted that the RVG-NLS- SEC nomenclature depicted in the title of the Figures correspond to G-N-SEC1749 as described herein. Specifically, G represents a rabies virus glycoprotein and N is a nuclear localization signal (NLS) amino acid sequence.

[0044] Figure 18 illustrates the effect of histones on the relative expression of

SEC1749 in N2A cells treated with unmodified SEC1749.

[0045] Figure 19 illustrates the effect of histones on the relative expression of

SEC1749 in N2A cells treated with the G-N-SEC1749 conjugate.

[0046] Figure 20 illustrates the effect of G-N conjugated to SEC on expression of

1749 in N2A cells.

[0047] Figure 21 illustrates BACE1 suppression in N2A cells following treatment with SEC1749 and G-N-SEC1749, with or without histones, as determined by a measurement of BACE1 expression relative to controls.

[0048] Figure 22 illustrates the effect of histones on the relative expression of

SEC1749 in N2A cells treated with SEC1749 and G-N-SEC1749, with or without histones.

[0049] Figure 23 illustrates BACE1 suppression in N2A cells following treatment with SEC1749 and G-N-SEC1749, with or without histones, as determined by a measurement of BACE1 expression relative to controls.

[0050] Figure 24 shows a comparison of SEC 1749 and mBACEl expression in treated N2A cells. [0051] Figure 25 compares the expression of SEC 1749 shRNA in N2A cells with the number of SEC 1749 DNA copies.

[0052] Figure 26 illustrates the effect of histones on the relative expression of

SEC 1749 in N2A cells treated with unmodified SEC, with or without histones.

[0053] Figure 27 illustrates BACE1 suppression in N2A cells following treatment with unmodified SEC 1749 with histones.

[0054] Figure 28 shows a comparison of SEC 1749 shRNA and BACE1 expression in

N2A cells treated with unmodified SEC 1749 with or without histones.

[0055] Figure 29 compares the expression of SEC 1749 shRNA in N2A cells with the number of SEC 1749 DNA copies.

[0056] Figure 30 shows the effect of histones on the relative expression of SEC 1749 in N2A cells treated with the conjugate with or without histones.

[0057] Figure 31 illustrates BACE1 suppression in N2A cells following treatment with G-N-SEC1749 with histones.

[0058] Figure 32 shows a comparison of SEC 1749 shRNA and BACE1 expression in

N2A cells treated with G-N-SEC1749 with or without histones.

[0059] Figure 33 compares the expression of SEC 1749 shRNA in N2A cells with the number of SEC 1749 DNA copies.

DETAILED DESCRIPTION OF THE INVENTION

[0060] The present invention includes peptides and peptide-polynucleotide compositions, including peptide-polynucleotide conjugates, and methods using those compositions for the delivery of DNA encoding for short, hairpin RNA (shRNA) and for the treatment of neurological diseases. The present invention overcomes the known limitations by providing a means to deliver a single or infrequent dose of an RNA-interference-based therapy. This is accomplished by delivering molecules of DNA encoding for short, hairpin RNA (shRNA) with a similar effect as the comparable siRNA in terms of target gene suppression. shRNA transcripts use a vector introduced into cells and generally utilize a promoter to ensure that the shRNA is always expressed. Thus, shRNA transfection persists in the cells longer than siRNA and allows for less frequent treatment. If certain viral vectors, such as lentivirus or other retroviral vectors resulting in integration of the delivered DNA into one of cell's chromosomes, are used, then the delivered DNA can be passed on to daughter cells, which causes inheritance of gene silencing. Delivery of molecules of DNA through the vasculature using the rabies virus glycoprotein and into target cells and cell nuclei is accomplished in the present invention by the provision of novel peptide sequences and peptide-polynucleotide compositions or conjugates. The compositions of the invention are capable of increasing shRNA expression in target cells. Certain formulations may have the further effect of reducing gene expression. Experiments associated with the Examples herein demonstrate that the peptides of the invention perform these functions. The present invention also provides for novel peptides and peptide-polynucleotide compositions and conjugates incorporating a peptide signal that has the function of transporting the DNA into the cell nucleus once the DNA is inside the cell. Finally, the novel peptide-polynucleotide conjugates of the present invention also provide direct, covalent attachment of DNA to the peptide at controlled molecular locations.

DEFINITIONS

[0061] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the relevant art.

[0062] The articles "a" and "an" are used herein to refer to one or to more than one

(i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element. [0063] The term "comprising" includes, but is not limited to, whatever follows the word "comprising." Thus, use of the term indicates that the listed elements are required or mandatory but that other elements are optional and may or may not be present.

[0064] The term "consisting of includes and is limited to whatever follows the phrase the phrase "consisting of." Thus, the phrase indicates that the limited elements are required or mandatory and that no other elements may be present.

[0065] The phrase "consisting essentially of includes any elements listed after the phrase and is limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase indicates that the listed elements are required or mandatory but that other elements are optional and may or may not be present, depending upon whether or not they affect the activity or action of the listed elements.

[0066] "Risk" relates to the possibility or probability of a particular event occurring either presently or at some point in the future. "Risk" can also refer to an assessment of known clinical risk factors that allows physicians and others of skill in the relevant art to classify patients from a low to high range of likelihood of developing a particular disease, disorder, or condition.

[0067] A "subject" or "patient" is a member of any animal species, preferably a mammalian species, optionally a human. The subject can be an apparently healthy individual, an individual suffering from a disease, or an individual being treated for a disease.

[0068] A "pharmaceutically effective" amount is that amount required to prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) of a disease state. The pharmaceutically effective amount depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, concurrent medication, and other factors that those skilled in the medical arts will recognize.

[0069] "Peptide-polynucleotide compositions" of the invention are compositions of matter in which the peptides of the invention are associated with the polynucleotides of the invention through the inclusion of a P domain in the peptide. The association may be, for example, an electrostatic association of the peptide with the polynucleotide.

[0070] "Peptide-polynucleotide conjugates" of the invention are peptide- polynucleotide compositions of the invention in which a polynucleotide of the invention is covalently linked to one or more peptides of the invention, and a P domain in the peptide or peptides is unnecessary and therefore optional, and preferably, omitted.

[0071] "RNA" means ribonucleic acid, a molecule consisting of ribonucleotides connected via a phosphate-ribose (sugar) backbone. "Ribonucleotide" means guanine, cytosine, uracil, or adenine or some nucleotide with a hydroxyl group at the 2 position of a beta-D- ribofuranose moiety. As is well known in the art, the genetic code uses thymidine as a base in DNA sequences and uracil in RNA. One skilled in the art knows how to replace thymidine with uracil in a written nucleic acid sequence to convert a written DNA sequence into a written RNA sequence, or vice versa.

[0072] By "gene," it is meant a region of DNA that controls the production of RNA.

In context of producing functional small interfering RNA (siRNA), this definition includes the necessary DNA sequence information encompassing the DNA sequences encoding the small interfering RNA, noncoding regulatory sequence and any included introns. The term "gene" is also meant to include a polynucleotide that includes a coding sequence or coding region. The present definition does not exclude the possibility that additional genes encoding proteins may function in association or in tandem with the genes encoding small interfering RNA. The gene may be of synthetic, cDNA or genomic origin, or a combination thereof. The gene may be one which occurs in nature, a non-naturally occurring gene which nonetheless encodes a naturally occurring polypeptide, or a gene which encodes a recognizable mutant of such a polypeptide. It may also encode an mRNA which will be "antisense" to a DNA found or an mRNA normally transcribed in the host cell, but which antisense RNA is not itself translatable into a functional protein.

[0073] "shRNA transcripts" are defined as short, hairpin RNA molecules that can be produced by a living cell upon being transfected or transduced from a nucleotide sequence encoding for the short, hairpin RNA. The sequence of the shRNA transcripts can be predicted from knowledge of the nucleotide sequence. Further, the shRNA transcripts can be detected in cell lysates using standard molecular biology techniques known to those skilled in the art.

[0074] "Messenger RNA" (mRNA) is defined as a single-stranded RNA molecule that is complementary to one of the DNA strands of a gene. The mRNA can be an RNA version of the gene that leaves the cell nucleus and moves to the cytoplasm where proteins are made. During protein synthesis, it is understood that an organelle called a ribosome moves along the mRNA, reads its base sequence, and uses the genetic code to translate each three-base triplet, or codon, into its corresponding amino acid.

[0075] "siRNA" is defined herein as a double-stranded RNA molecule that can, for example, interfere with the expression of a specific gene. There is no particular limitation in the length of siRNA

[0076] "Click chemistry" is defined as a chemical reaction in which heteroatom links are formed through a catalyzed reaction that has a high thermodynamic driving force. An example of such a reaction is the copper catalyzed Huisgen 1,3-dipolar cycloaddition reaction. [0077] A "vector" means any virus, as well as any plasmid, cosmid, phage, binary vector or segment of nucleic acid in single- or double- stranded or circular form that may or may not be self-transmissible or mobilizable, and that can transform eukaryotic host cells either by integration into the cellular genome or by existing extrachromosomally.

[0078] As used herein, the terms "target cell" or "target cells" refer to one or more cell, whether part of a multicellular or unicellular organism that is intended to receive the compositions or conjugates of the invention. The compositions and conjugates may be administered to the organism or host in a manner such that they are able to enter the target cell(s). Further, the cell can be a cell from an organism, a cell of an organism, a cell from a cell line, or a primary cell, in vivo or ex vivo (e.g., in cell culture).

[0079] As used herein, the term "transfection" means the introduction of a nucleic acid (e.g., an expression vector) into a recipient cell by non-viral methods generally, nucleic acid-mediated gene transfer, and the methods contemplated by the present invention.

[0080] "Transformation," as used herein, refers to a process in which a cell's genotype is changed as a result of the cellular uptake of exogenous DNA or RNA, and, for example, the transformed cell expresses a shRNA construct.

[0081] The phrase "expressing DNA" generally refers to transcription of DNA and, as appropriate, translation of the resulting mRNA transcript to a protein.

[0082] "Gene expression," as used herein, refers to the process by which information from a gene is used in the synthesis of a functional gene product. The products of gene expression can be proteins, but can also include non-functional RNA or other gene products.

[0083] "shRNA expression" refers to the transcription of the shRNA and, as appropriate, translation of the resulting mRNA transcript to a protein. Thus, as will be clear from the context, expression of a protein coding sequence results from transcription and translation of the coding sequence wherein "increasing shRNA expression" refers to greater transcription of the shRNA as compared to shRNA expression that has not been increased.

[0084] By "complementary strand," it is meant that a molecule comprised of one or more nucleic acids can form hydrogen bond(s) with another molecule comprised of one or more nucleic acids by either traditional Watson-Crick pairing or other non-traditional types.

[0085] "RNA interference," as defined herein, is a process that suppresses protein translation by either degrading the mRNA before it can be translated or by binding the mRNA and directly preventing its translation. This naturally-occurring mechanism of RNA interference can also be artificially induced to occur in cells. For example, RNA interference can be achieved by introducing into cells short, double- stranded nucleic acid oligoribonucleotides (siRNA) complementary to the mRNA for the gene to be suppressed or by introducing into cells a sequence of DNA that encodes for a short, hairpin transcript of nucleic acids (shRNA) that folds back upon itself and forms a short, double- stranded nucleic acid oligoribonucleotide that requires further processing in the cell.

[0086] "Histones" are highly alkaline proteins found in eukaryotic cell nuclei, which package and order the DNA into structural units called nucleosomes. They are the chief protein components of chromatin and act as spools around which DNA winds.

[0087] A "catheter" in communication with a drug delivery device can be an intraarterial or intravenous catheter, or a catheter specially adapted for insertion into an artery or vein. The catheter is generally a small tube configured for delivering a fluid, and is usually introduced through a small incision into the bodily vessel, channel, canal, or chamber in question; or into a bodily vessel, channel, canal, or chamber that is otherwise connected to the site of interest (or target site), and then guided through that vessel to the target site. [0088] By the phrase, "in communication," it is meant that the elements of the system of the invention are so connected by mechanical, fluid, or electrical contact so that a drug, fluid, data and/or instructions can be communicated among and between the elements.

[0089] The phrases, "intracarotid administration," "intracarotid artery delivery," and

"intracarotid dosing" refer to delivery, by injection, infusion, catheterization, or delivery of a substance via the carotid artery of a patient by means known to those of skill in the art.

[0090] "Femoral administration" refers to delivery, by injection, infusion, catheterization, or delivery of a substance via the femoral artery of a patient by means known to those of skill in the art.

[0091] "Drug delivery device" encompasses any and all devices that administers a therapeutic agent to a patient and includes infusion pumps, implanted or percutaneous vascular access ports, direct delivery catheter systems, local drug-release devices or any other type of medical device that can be adapted to deliver a therapeutic to a patient.

[0092] The terms "delivering," "deliver," "administering," and "administers" can be used interchangeably to indicate the introduction of a therapeutic or diagnostic agent into the body of a patient in need thereof to treat a disease or condition, and can further mean the introduction of any agent into the body for any purpose.

[0093] A "buffer solution" is an aqueous solution consisting of a mixture of a weak acid and its conjugate base or a weak base and its conjugate acid. It has the property that the pH of the solution changes very little when a small amount of strong acid or base is added to it. Buffer solutions are used as a means of keeping pH at a nearly constant value in a wide variety of chemical applications.

[0094] "Pharmaceutically acceptable buffer" is meant to encompass any buffer, which does not interfere with the effectiveness of the biological activity of the active ingredient and that is not toxic to the host to which it is administered. [0095] Administration "intracranially" refers to delivery of an agent being delivered into the cranial cavity, or intracranial space, of the subject, which refers to the space inside the skull.

[0096] Administration "intraparenchymally" refers to delivery of an agent into the parenchyma of an organ, which is tissue that constitutes the essential part of an organ, as distinguished from its supportive framework.

[0097] A G peptide is a molecule consisting of an amino acid chain wherein the sequence of amino acids corresponds to the glycoprotein domain of the rabies virus glycoprotein. Specifically, the sequence of amino acids corresponding to the glycoprotein domain ("G domain") is as follows, using the single letter code for amino acids as is recognized by those skilled in the art, reading left-to-right from the amino terminal end to the carboxyl terminal end:

SEQ ID No. 1 : YTIWMPEX₁PRX₂X₃X₄X₅CDIX₆TNSRGKRASN where is N or D; X₂ is P or L, X₃ is G or R, X₄ is T or P, X₅ is P or S, and X₆ is F or L. A preferred embodiment of the G peptide is used in the description and examples of the peptide and peptide-polynucleotide compositions and conjugates of the invention and has the following sequence:

SEQ ID No. 81 : YTr MPENPRPGTPCDIFTNSRGKRASN However, it is to be understood that the compositions, conjugates, and methods of the invention containing and utilizing the G peptide are not limited to this preferred embodiment but may be composed of any of the G peptides encompassed by SEQ ID No. 1.

[0098] A P domain of a peptide of the invention is defined as a sequence of amino acids that function to associate with DNA. For example, the P domain may correspond to protamine, as follows:

SEQ ID No. 2: RSQSRSRYYRQRQRSRRRRRRS Alternatively, the P domain may be comprised of another sequence of amino acids that function to associate with DNA, such as poly-lysine. For example, the P domain may be a sequence of 5-100 amino acids corresponding to poly-lysine, such as the following sequence of 9 lysines:

SEQ ID No. 31: KKKKKKKKK

[0099] An N domain of a peptide of the invention is defined as a sequence of amino acids corresponding to a nuclear localization signal (NLS), examples of which are as follows:

SEQ ID No. 5: PKKKRKV

SEQ ID No. 64: KKKNQQLKVGILHLGSRQKK

SEQ ID No. 65: KRPAATKKAGQAKKKK

[00100] A "Silencer Expression Cassette" (SEC) is defined as any sequence of DNA having a promoter sequence, a sequence encoding for a short, hairpin RNA (shRNA) transcript, and a transcription termination sequence. The promoter sequence may be (1) any DNA sequence comprising an RNA Polymerase II promoter, such as the cytomegalovirus promoter, the neuron specific enolase promoter, the chicken beta actin promoter, etc., or (2) any DNA sequence comprising an RNA Polymerase III promoter, such as the U6 promoter, the HI promoter, etc. The sequence coding for an shRNA transcript is any DNA sequence comprised of 19 to 29 bases encoding for RNA, and preferably, 19 to 21 bases encoding for RNA, followed by a loop sequence comprised of six to 20 bases of DNA, and followed by the DNA sequence comprised of the reverse complement of said 19 to 29 DNA bases, or preferably, 19 to 21 bases, encoding for RNA. The transcription termination sequence is six bases of deoxythymidine signaling termination of transcription to an RNA Polymerase III (Sequence TTTTTT), or a DNA sequence signaling termination of transcription to an RNA Polymerase II. A non-limiting example of a Silencer Expression Cassette is provided by the sequence named "SEC1749 basic," specified in SEQ ID No. 11, which encodes a U6 promoter, a sequence of bases encoding for RNA targeting BACEl (beta amyloid cleaving enzyme type 1), a loop sequence, the sequence of bases encoding for the reverse complement of the RNA targeting BACEl, and a transcription termination sequence of six bases of deoxythymidine. The DNA sequences of SEC 1749 basic have previously been disclosed in U.S. Patent Application Publication No. 2004/0220132 as "MB1749" and are incorporated herein by reference. Methods of making and using DNA encoding shRNA have been disclosed in at least U.S. Patent Application Publication Nos. 2004/0162255, 2004/0220132, and in U.S. Patent No. 7,618,948, the contents of which are hereby incorporated by reference. In particular, U.S. Patent Application Publication No. 2004/0220132, paragraphs [0150]- [0153], and U.S. Patent 7,618,948 each describe how to construct SEC1749 basic, which consists of DNA encoding for shRNA targeting BACEl that is generated from MB 1749. SEQ ID No. 11: 5' - ggatccgacgccgccatctctaggcccgcgccggccccctcgcacagacttgtgggagaa gctcggctactcccctgccccggttaatttgcatataatatttcctagtaactatagagg

cttaatgtgcgataaaagacagataatctgttctttttaatactagctacattttacatg

ataggcttggatttctataagagatacaaatactaaattattattttaaaaaacagcaca

aaaggaaact caccctaact gtaaagtaat tgtgtgtttt gagactataa atatcccttg gagaaaagccttgtttgggccgaagactgtggctacaacattcttcaagagagaatgttg tagccacagtcttctttttt - 3'

Another non-limiting example of a Silencer Expression cassette is used in the Examples and is provided by the sequence named "SEC1749," specified in SEQ ID No. 30, which also encodes for shRNA targeting BACEl and is comprised of a plasmid sequence fragment from pSilencer-1.0-U6 (Ambion, Inc.), SEC 1749 basic, and a second plasmid sequence fragment from pSilencer-1.0-U6. SEQ ID No. 30: 5' - cgcgcgtaatacgactcactatagggcgaattgggtacccgctctagaactagtggatcc gacgccgccatctctaggcccgcgccggccccctcgcacagacttgtgggagaagctcgg ctactcccctgccccggttaatttgcatataatatttcctagtaactatagaggcttaat

gtgcgataaaagacagataatctgttctttttaatactagctacattttacatgataggc

ttggatttctataagagatacaaatactaaattattattttaaaaaacagcacaaaagga

aactcaccctaactgtaaagtaattgtgtgttttgagactataaatatcccttggagaaa

agccttgtttgggccgaagactgtggctacaacattcttcaagagagaatgttgtagcca cagtcttctt ttttgaattcctgcagcccgggggatccactagttctagagcggccgcca

ccgcggtggagctccagcttttgttccctttagtgagggttaattgcgcg - 3'

[00101] A GP peptide of the invention is a molecule consisting of an amino acid chain wherein the sequence of amino acids is comprised of two domains. The first domain is the sequence of amino acids corresponding to the glycoprotein (G) domain of the rabies virus glycoprotein. (SEQ ID No. 1). The second domain is a sequence of amino acids corresponding to the P domain, as in SEQ ID Nos. 2 and 31. In a GP peptide, the first domain precedes the second domain, reading from the amino terminal end to the carboxyl terminal end, as follows, for example:

SEQ ID No. 3: YTIWMPENPRPGTPCDIFTNSRGKRASN

RSQSRSRYYRQRQRSRRRRRRS

Optionally, a GP peptide can also include a linker consisting of 1 to 100 glycines between the carboxyl end of the G domain and the amino end of the P domain, as follows, for example having 4 glycines:

SEQ ID No. 4: YTr MPENPRPGTPCDIFTNSRGKRASNGGGG

RSQSRSRYYRQRQRSRRRRRRS

Optionally, a GP peptide can also include an amino acid derivative with azide or alkyne functionality. This amino acid derivative can be located anywhere along the amino acid chain. An example of an azide functional GP peptide has the following sequence, which contains a linker having 4 consecutive glycines followed by K* and then followed by 3 glycines:

SEQ ID No. 28: YTr MPENPRPGTPCDIFTNSRGKRASNGGGGK*GGG

RSQSRSRYYRQRQRSRRRRRRS where K* is a lysine whose side chain amine has been reacted with NHS-(PEO)4-azide, such that the modified side chain has a poly(ethyleneoxdie) (otherwise known as polyethylene glycol (PEG)) with a terminal azide group, as in the formulae: H

Lysine derivatized with PEG-azide

Alternatively, an arginine amino acid could be modified in this way. The number of PEG repeat groups on this linker between the polynucleotide and peptide can be adjusted from 1 to 150, depending on the desired properties such as particle size, solubility and immunogenicity of the peptide-polynucleotide composition.

[00102] A PG peptide is a molecule consisting of an amino acid chain wherein the sequence of amino acids is comprised of two domains. The first domain is a sequence of amino acids corresponding to the P domain as in SEQ ID Nos. 2 and 31. The second domain is the sequence of amino acids corresponding to the glycoprotein (G) domain of the rabies virus glycoprotein (SEQ ID No. 1). In a PG peptide, the first domain precedes the second domain, reading from the amino terminal end to the carboxyl terminal end, as follows, for example:

SEQ ID No. 12: RSQSRSRYYRQRQRSRRRRRRS

YTr MPENPRPGTPCDIFTNSRGKRASN Optionally, a PG peptide can also include a linker consisting of 1 to 100 glycines between the carboxyl end of the P domain and the amino end of the G domain, as follows, for example having 4 glycines:

SEQ ID No. 19: RSQSRSRYYRQRQRSRRRRRRSGGGG

YTIWMPENPRPGTPCDIFTNSRGKRASN

Optionally, a PG peptide can also include an amino acid derivative with azide or alkyne functionality. This amino acid derivative can be located anywhere along the amino acid chain. An example of an azide functional PG peptide has the following sequence, which contains a linker having 4 consecutive glycines followed by K* and then followed by 3 glycines:

SEQ ID No. 29: RSQSRSRYYRQRQRSRRRRRRSGGGGK*GGG

YTIWMPENPRPGTPCDIFTNSRGKRASN where K* is a lysine whose side chain amine has been reacted with NHS-(PEO)4-azide, such that the modified side chain has a poly(ethyleneoxdie) (otherwise known as polyethylene glycol (PEG)) with a terminal azide group, as in the formulae:

H

Lysine derivatized with PEG-azide Alternatively, an arginine amino acid could be modified in this way. The number of PEG repeat groups on this linker between the polynucleotide and peptide can be adjusted from 1 to 150, depending on the desired properties such as particle size, solubility and immunogenicity of the peptide-polynucleotide composition.

[00103] A GPN peptide is a molecule consisting of an amino acid chain wherein the sequence of amino acids is comprised of three domains. The first domain is the sequence of amino acids corresponding to the glycoprotein (G) domain of the rabies virus glycoprotein

(SEQ ID No. 1). The second domain is a sequence of amino acids corresponding to the P domain, as in SEQ ID No. 2. The third domain is a sequence of amino acids corresponding to a nuclear localization signal (N domain), as in SEQ ID Nos. 5 and 64-65. An example of a

GPN peptide sequence is as follows:

SEQ ID No. 6: YTIWMPENPRPGTPCDIFTNSRGKRASN

RSQSRSRYYRQRQRSRRRRRRSPKKKRKV

Optionally, a GPN peptide can also include two linkers consisting of (1) 1 to 100 glycines between the carboxyl end of the G domain and the amino end of the P domain and (2) 1 to

100 glycines between the carboxyl end of the P domain and the amino end of the N domain.

The following example has a linker of 4 glycines located between G and P and a linker of 3 glycines located between P and N:

SEQ ID No. 7: YTr MPENPRPGTPCDIFTNSRGKRASNGGGG RSQSRSRYYRQRQRSRRRRRRSGGGPKKKRKV

[00104] A PNG peptide is a molecule consisting of an amino acid chain wherein the sequence of amino acids is comprised of three domains. The first domain is a sequence of amino acids corresponding to the P domain, as in SEQ ID Nos. 2 and 31. The second domain is a sequence of amino acids corresponding to a nuclear localization signal (N domain), as in

SEQ ID Nos. 5 and 64-65. The third domain is the sequence of amino acids corresponding to the glycoprotein (G) domain of the rabies virus glycoprotein (SEQ ID No. 1). An example of a PNG peptide is as follows:

SEQ ID No. 14: RSQSRSRYYRQRQRSRRRRRRSPKKKRKV

YTIWMPENPRPGTPCDIFTNSRGKRASN

Optionally, a PNG peptide can also include two linkers consisting of (1) 1 to 100 glycines between the carboxyl end of the P domain and the amino end of the N domain and (2) 1 to

100 glycines between the carboxyl end of the N domain and the amino end of the G domain.

The following example has a linker of 3 glycines located between P and N and a linker of 4 glycines located between N and G:

SEQ ID No. 8: RSQSRSRYYRQRQRSRRRRRRSGGGPKKKRKVGGGG

YTIWMPENPRPGTPCDIFTNSRGKRASN

[00105] A GN peptide is a molecule consisting of an amino acid chain wherein the sequence of amino acids is comprised of two domains. A GN peptide may also be described as a RVG-NLS peptide. The first domain is the sequence of amino acids corresponding to the glycoprotein (G) domain of the rabies virus glycoprotein. (SEQ ID No. 1). The second domain is a sequence of amino acids corresponding to a nuclear localization signal (N domain), such as SEQ ID Nos. 5 and 64-65. In a GN peptide, the first domain precedes the second domain, reading from the amino terminal end to the carboxyl terminal end, as follows, for example:

SEQ ID No. 15: YTr MPENPRPGTPCDIFTNSRGKRASNPKKKRKV Optionally, a GN peptide can also include a linker consisting of 1 to 100 glycines between the carboxyl end of the G domain and the amino end of the N domain, an example of which is as follows having 4 glycines:

SEQ ID No: 9: YTr MPENPRPGTPCDIFTNSRGKRASNGGGGPKKKRKV Optionally, a GN peptide can also include an amino acid derivative with azide or alkyne functionality. This amino acid derivative can be located anywhere along the amino acid chain. The azide functional GN peptide used in the Examples has the following sequence and contains a linker having 4 consecutive glycines followed by K* and then followed by 3 glycines:

SEQ ID No. 16: YTIWMPENPRPGTPCDIFTNSRGKRASNGGGGK*GGG PKKKRKV where K* is a lysine whose side chain amine has been reacted with NHS-(PEO)4-azide, such that the modified side chain has a poly(ethyleneoxdie) (otherwise known as polyethylene glycol (PEG)) with a terminal azide group, as in the formulae:

H

Lysine

Lysine derivatized with PEG-azide

Alternatively, an arginine amino acid could be modified in this way. The number of PEG repeat groups on this linker between the polynucleotide and peptide can be adjusted from 1 to 150, depending on the desired properties such as particle size, solubility and immunogenicity of the peptide-polynucleotide conjugate.

[00106] A NG peptide is defined as a molecule consisting of an amino acid chain wherein the sequence of amino acids is comprised of two domains. The first domain is amino acids corresponding to the N domain, such as SEQ ID Nos. 5 and 64-65. The second domain is the sequence of amino acids corresponding to the glycoprotein (G) domain of the rabies virus glycoprotein. (SEQ ID No. 1). In an NG peptide, the first domain precedes the second domain, reading from the amino terminal end to the carboxyl terminal end, as follows, for example:

SEQ ID No. 17: PKKKRKVYTIWMPENPRPGTPCDIFTNSRGKRASN Optionally, an NG peptide can also include a linker consisting of 1 to 100 glycines between the carboxyl end of the N domain and the amino end of the G domain, an example of which is as follows:

SEQ ID No: 10: PKKKRKVGGGGYTr MPENPRPGTPCDIFTNSRGKRASN Optionally, an NG peptide can also include an amino acid derivative with azide or alkyne functionality. This amino acid derivative can be located anywhere along the amino acid chain. A non-limiting example of an azide functional NG peptide has the following sequence and contains a linker having 3 consecutive glycines followed by K* and then followed by 4 glycines:

SEQ ID No. 18: PKKKRKVGGGK*GGGGYTr MPENPRPGTPCDIFTNSRGKRASN where K* is a lysine whose side chain amine has been reacted with NHS-(PEO)4-azide, such that the modified side chain has a poly(ethyleneoxdie) (otherwise known as polyethylene glycol (PEG)) with a terminal azide group, as in the formulae above. Alternatively, an arginine amino acid could be modified in this way. The number of PEG repeat groups on this linker between the polynucleotide and peptide can be adjusted from 1 to 150, depending on the desired properties such as particle size, solubility and immunogenicity of the peptide- polynucleotide conjugate.

[00107] A GNP peptide is a molecule consisting of an amino acid chain wherein the sequence of amino acids is comprised of three domains. The first domain is the sequence of amino acids corresponding to the glycoprotein (G) domain of the rabies virus glycoprotein (SEQ ID No. 1). The second domain is a sequence of amino acids corresponding to the N domain, such as SEQ ID Nos. 5 and 64-65. The third domain is a sequence of amino acids corresponding to the P domain, as in SEQ ID Nos. 2 and 31. An example of a PNG peptide is as follows:

SEQ ID No. 20: YTr MPENPRPGTPCDIFTNSRGKRASN

PKKKRKVRSQSRSRYYRQRQRSRRRRRRS

Optionally, a GNP peptide can also include two linkers consisting of (1) 1 to 100 glycines between the carboxyl end of the G domain and the amino end of the N domain and (2) 1 to

100 glycines between the carboxyl end of the N domain and the amino end of the P domain.

The following example contains a linker of 3 glycines located between G and N and a linker of 4 glycines located between N and P:

SEQ ID No. 21: YTr MPENPRPGTPCDIFTNSRGKRASNGGG PKKKRKVGGGGRSQSRSRYYRQRQRSRRRRRRS

[00108] A PGN peptide is a molecule consisting of an amino acid chain wherein the sequence of amino acids is comprised of three domains. The first domain is a sequence of amino acids corresponding to the protamine domain, as in SEQ ID Nos. 2 and 31. The second domain is the sequence of amino acids corresponding to the glycoprotein (G) domain of the rabies virus glycoprotein (SEQ ID No. 1). The third domain is a sequence of amino acids corresponding to the N domain, such as SEQ ID Nos. 5 and 64-65. An example of a

PGN peptide is as follows:

SEQ ID No. 22: RSQSRSRYYRQRQRSRRRRRRS

YTIWMPENPRPGTPCDIFTNSRGKRASNPKKKRKV

Optionally, a PGN peptide can also include two linkers consisting of (1) 1 to 100 glycines between the carboxyl end of the P domain and the amino end of the G domain and (2) 1 to

100 glycines between the carboxyl end of the G domain and the amino end of the N domain. The following example contains a linker of 4 glycines located between P and G and a linker of 4 glycines located between G and N:

SEQ ID No. 23: RSQSRSRYYRQRQRSRRRRRRSGGGG YTIWMPENPRPGTPCDIFTNSRGKRASNGGGGPKKKRKV

[00109] A NGP peptide is a molecule consisting of an amino acid chain wherein the sequence of amino acids is comprised of three domains. The first domain is a sequence of amino acids corresponding to an N domain, such as SEQ ID Nos. 5 and 64-65. The second domain is the sequence of amino acids corresponding to the glycoprotein (G) domain of the rabies virus glycoprotein (SEQ ID No. 1). The third domain is a sequence of amino acids corresponding to the P domain, as in SEQ ID Nos. 2 and 31. An example of a NGP peptide is as follows:

SEQ ID No. 24: PKKKRKVYTr MPENPRPGTPCDIFTNSRGKRASN

RSQSRSRYYRQRQRSRRRRRRS

Optionally, a NGP peptide can also include two linkers consisting of (1) 1 to 100 glycines between the carboxyl end of the N domain and the amino end of the G domain and (2) 1 to

100 glycines between the carboxyl end of the G domain and the amino end of the P domain.

The following example contains a linker of 4 glycines located between N and G and a linker of 4 glycines located between G and P.

SEQ ID No. 25: PKKKRKVGGGGYTr MPENPRPGTPCDIFTNSRGKRASN

GGGGRSQSRSRYYRQRQRSRRRRRRS

[00110] A NPG peptide is a molecule consisting of an amino acid chain wherein the sequence of amino acids is comprised of three domains. The first domain is a sequence of amino acids corresponding to the N domain, such as SEQ ID Nos. 5 and 64-65. The second domain is a sequence of amino acids corresponding to the P domain, as in SEQ ID Nos. 2 and

31. The third domain is the sequence of amino acids corresponding to the glycoprotein (G) domain of the rabies virus glycoprotein (SEQ ID No. 1). An example of a NPG peptide is as follows:

SEQ ID No. 26: PKKKRKVRSQSRSRYYRQRQRSRRRRRRS

YTIWMPENPRPGTPCDIFTNSRGKRASN

Optionally, a NPG peptide can also include two linkers consisting of (1) 1 to 100 glycines between the carboxyl end of the N domain and the amino end of the P domain and (2) 1 to

100 glycines between the carboxyl end of the P domain and the amino end of the G domain.

The following example contains a linker of 4 glycines located between N and P and a linker of 4 glycines located between P and G:

SEQ ID No. 27: PKKKRKVGGGGRSQSRSRYYRQRQRSRRRRRRS

GGGGYTr MPENPRPGTPCDIFTNSRGKRASN

[00111] A PEG peptide is a molecule consisting of amino acids including any of the previously described peptides having three domains wherein the sequence of amino acids is comprised of the three domains and also a moiety of polyethylene glycol (PEG). A PEG polymer that has a molecular weight in the range of 2,000 Da to 10,000 Da may be located anywhere along the amino acid chain. For example, in a PN-PEG-G peptide, a PNG peptide also contains this moiety of PEG. Here, the carboxyl end of the N chain is covalently coupled to one end of the polymer, and the other end of the polymer is covalently coupled to the amino end of the G domain. Thus, a PEG and/or a linker of 1 to 100 glycines in any order or combination thereof represented by variable X may exist in any of the following 24 permutations, examples of which are illustrated in the identified sequences:

PN-PEG-G

SEQ ID No. 39: RSQSRSRYYRQRQRSRRRRRRSPKKKRKVX

YTIWMPENPRPGTPCDIFTNSRGKRASN

PG-PEG-N

SEQ ID No. 40: RSQSRSRYYRQRQRSRRRRRRS

YTIWMPENPRPGTPCDIFTNSRGKRASNXPKKKRKV GP-PEG-N

SEQ ID No. 41: YTr MPENPRPGTPCDIFTNSRGKRASN

RSQSRSRYYRQRQRSRRRRRRSXPKKKRKV

NP-PEG-G

SEQ ID No. 42: YTrWMPENPRPGTPCDIFTNSRGKRASN

PKKKRKVXRSQSRSRYYRQRQRSRRRRRRS

NG-PEG-P

SEQ ID No. 43: PKKKRKVRSQSRSRYYRQRQRSRRRRRRSX

YTIWMPENPRPGTPCDIFTNSRGKRASN

GN-PEG-P

SEQ ID No. 44: PKKKRKVYTrWMPENPRPGTPCDIFTNSRGKRASNX

RSQSRSRYYRQRQRSRRRRRRS

N-PEG-GP

SEQ ID No. 45: PKKKRKVXYTrWMPENPRPGTPCDIFTNSRGKRASN

RSQSRSRYYRQRQRSRRRRRRS

N-PEG-PG

SEQ ID No. 46: PKKKRKVXRSQSRSRYYRQRQRSRRRRRRS

YT VMPENPRPGTPCDIFTNSRGKRASN

G-PEG-NP

SEQ ID No. 47: YTrWMPENPRPGTPCDIFTNSRGKRASNX

PKKKRKVRSQSRSRYYRQRQRSRRRRRRS

G-PEG-PN

SEQ ID No. 48: YTrWMPENPRPGTPCDIFTNSRGKRASNX

RSQSRSRYYRQRQRSRRRRRRSPKKKRKV

P-PEG-GN

SEQ ID No. 49: RSQSRSRYYRQRQRSRRRRRRSX

YTFWMPENPRPGTPCDIFTNSRGKRASNPKKKRKV

P-PEG-NG

SEQ ID No. 50: RSQSRSRYYRQRQRSRRRRRRSXPKKKRKV

YTrWMPENPRPGTPCDIFTNSRGKRASN GPN-PEG

SEQ ID No. 51: YTr MPENPRPGTPCDIFTNSRGKRASN RSQSRSRYYRQRQRSRRRRRRSPKKKRKVX

GNP-PEG

SEQ ID No. 52: YTrWMPENPRPGTPCDIFTNSRGKRASN PKKKRKVRSQSRSRYYRQRQRSRRRRRRSX

PNG-PEG

SEQ ID No. 53: RSQSRSRYYRQRQRSRRRRRRSPKKKRKV

YTIWMPENPRPGTPCDIFTNSRGKRASNX

PGN-PEG

SEQ ID No. 54: RSQSRSRYYRQRQRSRRRRRRS

YTIWMPENPRPGTPCDIFTNSRGKRASNPKKKRKVX

NPG-PEG

SEQ ID No. 55: PKKKRKVRSQSRSRYYRQRQRSRRRRRRS

YTIWMPENPRPGTPCDIFTNSRGKRASNX

NGP-PEG

SEQ ID No. 56: PKKKRKVYTrWMPENPRPGTPCDIFTNSRGKRASN

RSQSRSRYYRQRQRSRRRRRRSX

PEG-GPN

SEQ ID No. 57: XYTIWMPENPRPGTPCDIFTNSRGKRASN RSQSRSRYYRQRQRSRRRRRRSPKKKRKV

PEG-GNP

SEQ ID No. 58: XYTIWMPENPRPGTPCDIFTNSRGKRASN PKKKRKVRSQSRSRYYRQRQRSRRRRRRS

PEG-PNG

SEQ ID No. 59: XRSQSRSRYYRQRQRSRRRRRRSPKKKRKV

YTIWMPENPRPGTPCDIFTNSRGKRASN

PEG-PGN

SEQ ID No. 60: XRSQSRSRYYRQRQRSRRRRRRS

YTIWMPENPRPGTPCDIFTNSRGKRASNPKKKRKV PEG-NPG

SEQ ID No. 61: XPKKKRKVRSQSRSRYYRQRQRSRRRRRRS

YTIWMPENPRPGTPCDIFTNSRGKRASN

PEG-NGP

SEQ ID No. 62: XPKKKRKVYTr MPENPRPGTPCDIFTNSRGKRASN

RSQSRSRYYRQRQRSRRRRRRS

Notably, the PEG peptide can also contain a linker consisting of 1 to 100 glycines located between any domains not linked to PEG. For example, in the PN-PEG-G peptide, a linker of

1 to 100 glycines may be located between the carboxyl end of the P domain and the amino end of the N domain, as is illustrated by the following sequence:

SEQ ID No. 63: RSQSRSRYYRQRQRSRRRRRRSGGG PKKKRKVXYTr MPENPRPGTPCDIFTNSRGKRASN

PEPTIDE-POLYNUCLEOTIDE CONJUGATES

[00112] The DNA encoding for shRNA in the present invention are part of Silencer

Expression Cassettes. The transcription cassette preferably contains a promoter sequence, a sequence encoding for a shRNA transcript, and a transcription termination sequence. Promoters are regions of DNA that facilitate the transcription of a particular gene. A promoter is usually a sequence upstream (5') of the nucleotide sequence of interest, which directs and/or controls expression of the nucleotide sequence by providing for recognition by RNA polymerase and other factors required for proper transcription. The promoter sequence may be any DNA sequence comprising an RNA Polymerase II promoter, such as the following: the cytomegalovirus (CMV) promoter

SEQ ID No. 34: 5' - gcggccgcacgcgtggagctagttattaatagtaatcaattacggggtcattagttcata gcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgc ccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgtcaatag ggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtac atcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccg cctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacg tattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggat agcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgt tttgcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgca aatgggcggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccg tcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccgggaccg atccagcctc - 3' the cytomegalovirus immediate early region enhancer followed by chicken beta-actin promoter, collectively also known as the CBA promoter

SEQ ID No. 35: 5' - actagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacata acttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgac gtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaact gcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaa atggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgta ttagtcatcgctattaccatggtcgaggtgagccccacgttctgcttcactctccccatctcccccccct ccccacccccaattttgtatttatttattttttaattattttgtgcagcgatgggggcgggggggggggg ggggcgcgcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcggagag gtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcgg cggcggccctataaaaagcgaagcgcgcggcgggcgg - 3'

the neuron- specific enolase promoter

SEQ ID No. 36: 5' - cgcgcacgcacacacacacggaaggaggaaggaggagatggatgaacacatgcagggtcagag ggatttttctctaagctcctcttttctgacggacaattctttcaatttttttttgtggttctatcaatcaaaccca gggctgtgtgcatgtgaaatctgtaccccatcactgagcccaacacaacgccagtagagctcagagt gaggagtgctccttgaggcccagggcacctatggagatcagtctgcaatacttaacactggattcata aatgttcgaaaccacagagttttggaaagaagaacattacaagactgagctttttattcaagctggggg gctcaatccatccttagctctgggttccttactgaaggaagcactcccaccccacagtaccccactctt agctctgagctcctcctctgctcgcccaatccttccaaccccctatggtggtatggctgacacagaaa atgtctgctcctgtatgggacatttgcccctcttctccaaatataagacaggatgaggcctagcttttgct gctccaaagttttaaaagaacacattgcacggcatttagggactctaaagggtggaggaggaatgag ggaattgcatcatgccaaggctggtcctcatccatcactgcttccagggcccagagtggcttccagg aggtattcttacaaaggaagcccgatctgtagctaacactcagagcccattttcctgcgttaacccctc ccgacctcatatacaggagtaacatgatcagtgacctgggggagctggccaaactgcgggacctgc ccaagctgagggccttggtgctgctggacaacccctgtgccgatgagactgactaccgccaggag gccctggtgcagatggcacacctagagcgcctagacaaagagtactatgaggacgaggaccggg cagaagctgaggagatccgacagaggctgaaggaggaacaggagcaagaactcgacccggacc aagacatggaaccgtacctcccgccaacttagtggctcctctagcctgcagggacagtaaaggtgat ggcaggaaggcagcccccggaggtcaaaggctgggcacgcgggaggagaggccagagtcaga ggctgcgggtatctcagatatgaaggaaagatgagagaggctcaggaagaggtaagaaaagaca caagagaccagagaagggagaagaattagagagggaggcagaggaccgctgtctctacagacat agctggtagagactgggaggaagggatgaaccctgagcgcatgaagggaaggaggtggctggtg gtatatggaggatgtagctgggccagggaaaagatcctgcactaaaaatctgaagctaaaaataaca ggacacggggtggagaggcgaaaggagggcagattgaggcagagagactgagaggcctgggg atgtgggcattccggtagggcacacagttcacttgtct - 3'

RNA Polymerase II is an enzyme found in eukaryotic cells that catalyzes transcription of

DNA to synthesize precursors of mRNA and most SNO RNA and microRNA.

[00113] Alternatively, the promoter sequence may be any DNA sequence comprising an RNA Polymerase III promoter, such as the following:

The human U6 promoter

SEQ ID No. 32: 5' - tcgggcaggaagagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagag agataattagaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaata atttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagt atttcgatttcttggctttatatatcttgtggaaaggacgaaacacc - 3' the human HI promoter

SEQ ID No. 33: 5' - gaattcgaacgctgacgtcatcaacccgctccaaggaatcgcgggcccagtgtcactaggcggga acacccagcgcgcgtgcgccctggcaggaagatggctgtgagggacaggggagtggcgccctg caatatttgcatgtcgctatgtgttctgggaaatcaccataaacgtgaaatgtctttggatttgggaatctt ataagttccctatcagtgatag - 3'

RNA Polymerase III transcribes DNA to synthesized ribosomal 5S, rRNA, tRNA, and other small RNAs. The sequence coding for an shRNA transcript is any DNA sequence comprised of 19-29, and preferably, 19-21, bases encoding for RNA, followed by a loop sequence comprised of 6-20 bases of DNA, and further followed by the DNA sequence comprised of the reverse complement of the 19-29 or preferably 19-21 DNA bases encoding for RNA. The transcription termination sequence is six bases of deoxythymidine signaling termination of transcription to an RNA Polymerase III, or a DNA sequence signaling termination of transcription to an RNA Polymerase II.

[00114] Viral vectors have been widely used in gene therapy to deliver nucleotides to target cells. It is well known that a peptide derived from the rabies virus glycoprotein (RVG) interacts specifically with the nicotinic acetylcholine receptor (nAchR) on neural cells to enable viral entry. Because neurotropic viruses such as the rabies virus cross the blood-brain barrier to infect brain cells, they may be used to enter the central nervous system and deliver gene therapy transcripts to the brain. The blood-brain barrier serves a neuroprotective function by hindering the delivery of agents to the brain. In the past, craniotomy or intracerebral injection of therapeutic agents has been performed due to the inability of compositions containing these agents to effectively cross the blood-brain barrier. These delivery methods are highly invasive and do not allow for delivery of exogenous genes to all of the brain from a single infusion site. Kumar et al., supra, suggested that RVG peptides can be used for a delivery of siRNA into the brain across the blood-brain barrier by electrostatically associating the RVG peptide with the siRNA transcript. Thus, RVG peptide compositions can be administered intracranially or by injection into the carotid or femoral artery, by intraparenchymal infusion, or via an implanted drug delivery pump and achieve transfection of neural cells with the therapeutic nucleotide molecule.

[00115] Effectiveness of therapy using DNA encoding for shRNA relies upon the ability of the DNA to reach a target cell and its nucleus. The present invention includes a peptide composition that adds extensions to the RVG amino acid sequence to enable it to effectively deliver non-viral DNA expression cassettes to neurons throughout the brain. One extension is an amino acid domain consisting of a peptide sequence that has DNA condensing properties, which allows the resulting combined peptide to serve as a DNA carrier. An example of this extension is a protamine (P) domain having DNA condensing properties, as in SEQ ID No. 2. Alternatively, the P domain may be comprised of another sequence of amino acids that function to associate with DNA, such as poly-lysine, an example of which is SEQ ID No. 31. The protein resulting from the fusion of the RVG peptide and the first extension is a GP peptide or a PG peptide.

[00116] A second possible extension is an amino acid domain consisting of a peptide sequence that functions as a nuclear localization signal (NLS), such as SEQ ID Nos. 5 and 64-65. A peptide containing the NLS extension has the ability to deliver the DNA into the cell nucleus. The peptide resulting from the fusion of the RVG peptide with the second domain is a GN or NG peptide. The peptides resulting from the fusion of the RVG sequence with both extensions include GNP peptides, GPN peptides, PGN peptides, PNG peptides, NGP peptides, and NPG peptides, as previously defined. Any of the fused domains may be separated by an optional linker composed of up to 100 glycine amino acids. The complete, three-domain peptide consisting of a fusion of the RVG sequence with both the DNA condensing sequence and the NLS sequence enables transport of DNA into neurons, with subsequent intracellular escape from endosomes and transport of the DNA to the nuclear compartment, which is required for DNA utilization. The peptide compositions of the present invention provide novel formulations useful for widespread delivery of therapeutic DNA throughout target mammalian tissues.

[00117] Optionally, the peptide compositions of the present invention also contain a moiety of polyethylene glycol (PEG). This moiety is preferably a polyethylene glycol polymer (PEG) that has a molecular weight in the range of 2,000 Da to 10,000 Da, and it is covalently coupled to one of the amino acid domains at any location in the peptide chain. Covalent bonding further provides a stable composition resulting in efficient and predictable delivery of the polynucleotide.

[00118] The peptide composition of the invention may also contain an amino acid derivative having azide or alkyne functionality. This amino acid derivative may be at any location in the peptide chain. An azide functional group may be a PEG molecule having a terminal azide group where the number of PEG repeat groups is from 1 to 150. A non- limiting example of an amino acid derivative having azide functionality is a lysine whose side chain amine has been reacted with NHS-(PEO)₄-azide, so that the modified side chain has the described PEG molecule. This is the azide functional group used in the azide functional GN peptide used in the examples. Another example of an azide functional amino acid derivatives includes the amino acid analog azidohomoalanine. Additional azide or alkyne functional groups include, for example, azidoalanine, 2-amino-5-hexanoic acid, azidophenylalanine, or alkyl tyrosine derivative.

[00119] The peptide-polynucleotide conjugates of the present invention consist of the peptide sequences described above that are chemically linked to the therapeutic nucleotide, such as single- or double- stranded DNA, single- or double- stranded RNA, or associated with a plasmid. The polynucleotides may also be in the form of a SEC. The following description identifies specific peptide-polynucleotide conjugates encompassed by the invention. [00120] A GN-SEC conjugate is the molecular product of a chemical reaction whereby a GN peptide is covalently linked to the 5 prime (5') or 3 prime (3') end of a single- stranded or double-stranded DNA molecule encoding for a Silencer Expression Cassette. Optionally, a GN-SEC conjugate can be the molecular product of a chemical reaction whereby a GN peptide is covalently linked to the 5' end of a double- stranded DNA molecule encoding for a Silencer Expression Cassette and another GN peptide is covalently linked to the 5' end of the opposite strand of the same double- stranded DNA molecule. Optionally, a GN-SEC conjugate can be the molecular product of a chemical reaction whereby a GN peptide is covalently linked to the 3' end of a double- stranded DNA molecule encoding for a Silencer Expression Cassette and another GN peptide is covalently linked to the 3' end of the opposite strand of the same double- stranded DNA molecule. The covalent linkage of the GN peptide to the DNA molecule can be at any amino acid in the GN peptide sequence. A non-limiting example is a GN-SEC conjugate in which the 5' end of the DNA molecule has been covalently linked to a lysine amino acid (K*) that was modified to have an azide group on the side chain and was put into a series of glycine amino acids located between the carboxyl end of the G domain of the GN peptide and the amino end of the N domain of the GN peptide when the peptide was synthesized.

[00121] A NG-SEC conjugate is the molecular product of a chemical reaction whereby an NG peptide is covalently linked to the 5' or 3' end of a single-stranded or double- stranded DNA molecule encoding for a Silencer Expression Cassette. Optionally, an NG-SEC conjugate can be the molecular product of a chemical reaction whereby a NG peptide is covalently linked to the 5' end of a double- stranded DNA molecule encoding for a Silencer Expression Cassette and another NG peptide is covalently linked to the 5' end of the opposite strand of the same double-stranded DNA molecule. Optionally, a NG-SEC conjugate can be the molecular product of a chemical reaction whereby a NG peptide is covalently linked to the 3' end of a double- stranded DNA molecule encoding for a Silencer Expression Cassette and another NG peptide is covalently linked to the 3' end of the opposite strand of the same double-stranded DNA molecule. The covalent linkage of the NG peptide to the DNA molecule can be at any amino acid in the NG peptide sequence. A non-limiting example is a GN-SEC conjugate in which the 5' end of the DNA molecule has been covalently linked to a lysine amino acid that was modified to have an azide group on the side chain and was put into a series of glycine amino acids located between the carboxyl end of the N domain of the NG peptide and the amino end of the G domain of the NG peptide when the peptide was synthesized.

[00122] The chemical linkage between the peptide and the polynucleotide may be a covalent bond obtained through "click chemistry." Kolb H.C., Finn M.G., Sharpless K.B., Click Chemistry: Diverse Chemical Function from a Few Good Reactions, ANGEW. CHEM. INT. ED. ENGL., Jun. 1 , 2001 ; 40( 1 1): 2004-21). The general concept of "click chemistry" is to create heteroatom links through a reaction that is "spring loaded" with a high thermodynamic driving force. "Click chemistry" is highly specific, and there is little chance of undesirable side products. It provides very high yields, and it can be performed with simple reaction conditions, e.g., in water. Several different reactions fit under the "click chemistry" concept; the most commonly used is a reaction of an azide with an alkyne to form a five-membered heterocycle (triazole ring) using a copper catalyst.

[00123] Others have used "click chemistry" to label DNA with fluorophores (Seo T.S., et al., Click chemistry to construct fluorescent oligonucleotides for DNA sequencing, J. ORG. CHEM., Jan. 24, 2003; 68(2): 609- 12); to conjugate DNA to lipids (Godeau G., et al, Lipid- conjugated oligonucleotides via click chemistry efficiently inhibit Hepatitis C virus translation, J. MED. CHEM., Aug. 14, 2008; 51 ( 15): 4-6, e-pub Jul. 8, 2008); to conjugate the C-terminus of proteins to the 5' end of single-stranded DNA to form protein-DNA nanostructures (Duckworth B., et al., A universal method for the preparation of covalent protein-DNA conjugates for use in creating protein nanostructures, ANGEW. CHEM., 2007; 119: 8975-78); and to conjugate cell penetrating peptides to short polynucleic acid (PNA) and DNA strands (Gogoi, K., et al., A versatile method for the preparation of conjugates of peptides with DNA/PN A/analog by employing chemo-selective click reaction in water, NUCL. ACIDS RES., 2007; 35(21): el39, Epub Nov. 2, 2007).

[00124] Optionally, any of the peptide-polynucleotide compositions of the invention may be further combined with histones that condense the DNA of the peptide-polynucleotide composition. The histones are present in a molar ratio from 1: 1 histones to DNA to 31: 1 histones to DNA, or, generally, from n: l histones to DNA, for each n={x £ M| l < x < 31}. In certain embodiments, the ratio of histones to DNA include molar ratios of 6: 1, 15: 1, 30: 1, and 31: 1 histones to DNA.

[00125] The present invention includes novel methods of making peptide- polynucleotide conjugates wherein the "click chemistry" reaction is used to conjugate the 5' or 3' ends of double- stranded DNA molecules that express shRNA to a specific amino acid between two or three functional peptide domains so that the DNA is able to enter target cells and the nucleus of the cell.

[00126] An embodiment of the method of synthesizing peptide-polynucleotide conjugates of the invention includes a click chemical reaction using GN peptide molecules that have been synthesized such that one lysine amino acid residue in a specific location of the GN peptide has been modified with NHS-(PEO)₄-azide to produce a PEG-azide group on the side chain. The location for the modified lysine amino acid residue can be, for example, between the carboxyl end of the G domain and the amino end of the N domain. Alternatively, it could be located at the carboxyl end of the N domain or at the amino end of the G domain. An example of the azide modified GN peptide of the invention is shown above in SEQ ID No. 16. To affect coupling of the GN peptide to the 5' end of the DNA molecule by "click chemistry," that end of the DNA is modified to have an alkyne group. The click chemistry reaction between the azide-functional peptide and alkyne functional DNA is shown schematically in Figure 14. A double- stranded DNA molecule with an alkyne modification on the 5' end of one or both strands can be generated by synthesizing primers by polymerase chain reaction (PCR) using an alkyne functional phosphoramidite, such as 5'- hexynyl phosphoramidite, and then using those primers in the PCR synthesis of the double- stranded DNA molecule.

[00127] A non- limiting example of an alkyne modified DNA molecule is shown in

SEQ ID No. 38, which is SEC 1749 basic modified with an alkyne functional pho sphoramidite : SEQ ID No. 38: 5' -

G*GATCCGACGCCGCCATCTCTAGGCCCGCGCCGGCCCCCTC

GCACAGACTTGTGGGAGAAGCTCGGCTACTCCCCTGCCCCG

GTTAATTTGCATATAATATTTCCTAGTAACTATAGAGGCTTA

ATGTGCGATAAAAGACAGATAATCTGTTCTTTTTAATACTAG

CTACATTTTACATGATAGGCTTGGATTTCTATAAGAGATACA

AATACTAAATTATTATTTTAAAAAACAGCACAAAAGGAAAC

TCACCCTAACTGTAAAGTAATTGTGTGTTTTGAGACTATAAA

TATCCCTTGGAGAAAAGCCTTGTTTGGGCCGAAGACTGTGGC

TACAACATTCTTCAAGAGAGAATGTTGTAGCCACAGTCTTCT

TTTTT -3' where G* represents the attachment of the phosphoramidite.

In this example, the side chain on the azide modified amino acid of the peptide (K*) has the following structure:

The 5' end of the alkyne modified DNA molecule SEC1749 basic has the following structure:

Accordingly, the peptide-SEC conjugate of the modified peptide-SEC 1749 basic conjugate has the following structure:

The PCR methods used are well-known to those skilled in the art. A preferred embodiment of the method of synthesizing peptide-polynucleotide conjugates of the invention is illustrated in Example 9 below.

[00128] The compositions of the present invention are suitable for use in a method of transfecting a target cell with a polynucleotide encoding shRNA and methods of increasing shRNA expression in a target cell of a mammal. The method includes administering a peptide-polynucleotide composition or conjugate of the invention to a mammal, thereby causing increased shRNA expression in the target cell. The compositions are, in certain embodiments, combined with histones in a molar ratio from 1: 1 histones to DNA to 31: 1 histones to DNA, or, generally, from n: l histones to DNA, for each n={x £ ]R| l < x < 31}. Some embodiments of the ratio of histones to DNA include molar ratios of 6: 1, 15: 1, 30: 1, and 31: 1 histones to DNA. The compositions are preferably administered intracranially, intraparenchymally, by injection to the carotid or femoral artery, or intravenously. In one embodiment, the method of intracranial or intraparenchymal administration is by injection or by a catheter in communication with a drug delivery pump.

[00129] The invention also includes methods of suppressing gene expression in target cells of a mammal. In the method, the cells are transfected with any of the peptide- polynucleotide compositions of the invention, or naked siRNA. Preferably, the target genes are those associated with a neurological disease. As before, the compositions may be combined with histones in a molar ratio from 1 : 1 histones to DNA to 31: 1 histones to DNA, or, generally, from n: l histones to DNA, for each n={x £ M| l < x < 31}.

[00130] The compositions and methods of the present invention are suitable for use in the treatment of neurodegenerative diseases such as Huntington's disease, Alzheimer's disease, and Parkinson's disease. The DNA used in the invention for the peptide- polynucleotide compositions may comprise a SEC that includes a sequence encoding for a shRNA transcript targeting a gene or genes associated with a specific neurological disease. The shRNA transcripts that target the mRNA transcribed from the gene corresponding to a certain disease can prevent or delay symptoms of the disease by suppressing expression of associated proteins. For example, an appropriate target gene for Alzheimer's disease is beta- amyloid cleaving enzyme 1 (BACE1), including variants thereof, e.g., A, B, C, and D. The following table includes a non-exhaustive list of target genes for some known neurodegenerative diseases for which delivery of an SEC using the methods of the invention are expected to be therapeutic.

TABLE 1

[00131] As is well known to those skilled in the art, the DNA sequence for a Silencer

Expression Cassette encoding for shRNA targeting a gene may be constructed in a plasmid. The gene targets may include any of those listed in Table 1 or any other gene for which reduction of the expression of the corresponding gene product in the brain of a patient is desired. The construction of the SEC is performed by inserting the appropriate gene- specific shRNA sequence, a loop sequence, the reverse complement of the gene- specific shRNA sequence, and an appropriate termination sequence operably after a promoter sequence, such as the U6, HI, or cytomegalovirus (CMV) promoter. Various plasmids and instructions for their use in constructing shRNA are available from various commercial sources as the starting materials for making shRNA encoding plasmids, such as pSilencer™-2.10U6-neo, pSilencer™-3.1.-Hl-neo, and pSilencer™-4.1-CMV-hygro, which are each sold by Invitrogen, Inc. (Carlsbad, CA, USA). The linear, double- stranded Silencer Expression Cassette DNA molecule constructed in the plasmid can be recovered from the plasmid by cutting out the desired portion of the DNA molecule (the promoter, shRNA, loop, shRNA complement, and terminator) from the remainder of the plasmid using restriction enzymes. Alternatively, a quantity of the linear double-stranded Silencer Expression Cassette DNA molecule can be generated from the plasmid by a polymerase chain reaction (PCR) using primers that bracket the Silencer Expression Cassette region at the 5' and 3' ends in the plasmid.

[00132] Example SECs contemplated by the invention containing shRNA transcripts targeting genes listed in Table 1 are described as follows. SEC 1749 basic and SEC 1749 are as previously described (SEQ ID Nos. 11, 30, and 38). SEC1749 basic (SEQ ID No. 11) consists of a murine U6 promoter, the MB 1749 molecule, a loop, the reverse complement of the MB 1749 molecule, and a RNA Polymerase III transcription termination sequence (TTTTTT). The elements of the sequence are shown as follows:

SEQ ID No. 11: 5'

g

murine U6 promoter sequence gatccgacgccgccatctctaggcccgcgccggccccctcgcacagacttgtgg (SEQ ID No. 72) gagaagctcggctactcccctgccccggttaatttgcatataatatttcctagtaact atagaggcttaatgtgcgataaaagacagataatctgttctttttaatactagctacat tttacatgataggcttggatttctataagagatacaaatactaaattattattttaaaaa acagcacaaaaggaaactcaccctaactgtaaagtaattgtgtgttttgagactata aatatcccttggagaaaagccttgtttg

enzyme fragment ggccg

MB 1749 aagactgtggctacaacattc

loop sequence ttcaagaga

reverse complement MB 1749 gaatgttgtagccacagtctt

RNA Polymerase III transcription ctttttt

termination sequence Alternatively, SEC 1749 suitable for use in humans contains the human U6 promoter sequence, as follows:

SEQ ID No. 73: 5'- human U6 promoter sequence tcgggcaggaagagggcctatttcccatgattccttcatatttgcatatacgataca (SEQ ID No. 32) aggctgttagagagataattagaattaatttgactgtaaacacaaagatattagtaca aaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttt taaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatat atcttgtggaaaggacgaaacacc

MB 1749 aagactgtggctacaacattc

loop sequence ttcaagaga

reverse complement MB 1749 gaatgttgtagccacagtctt

termination sequence ctttttt

-3'

[00133] Another example of an SEC encoding shRNA targeting BACE1 corresponds to the BACE1 region surrounding nucleotide 1131 in the coding sequence for (mouse) BACE, described here as MB 1131 and provided in the following sequence:

SEQ ID No. 71: 5'- cgcgcgtaatacgactcactatagggcgaattgggtacccgctctagaactagtg murine U6 promoter sequence gatccgacgccgccatctctaggcccgcgccggccccctcgcacagacttgtgg gagaagctcggctactcccctgccccggttaatttgcatataatatttcctagtaact atagaggcttaatgtgcgataaaagacagataatctgttctttttaatactagctacat tttacatgataggcttggatttctataagagatacaaatactaaattattattttaaaaa acagcacaaaaggaaactcaccctaactgtaaagtaattgtgtgttttgagactata aatatcccttggagaaaagccttgtttg

enzyme fragment ggccg

MB1131 tgatcattggaggtatcga

loop sequence ttcaagaga

reverse complement MB 1131 tcgatacctccaatgatca

termination sequence ctttttt

gaattcctgcagcccgggggatccactagttctagagcggccgccaccgcggtg gagctccagcttttgttccctttagtgagggttaattgcgcg

-3'

Accordingly, the counterpart of this SEC suitable for human use (using a human promoter sequence as shown) is as follows: SEQ ID No. 80:

human U6 promoter sequence tcgggcaggaagagggcctatttcccatgattccttcatatttgcatatacgataca aggctgttagagagataattagaattaatttgactgtaaacacaaagatattagtaca aaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttt taaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatat atcttgtggaaaggacgaaacacc

BACEl-1131 tgatcattggaggtatcga

loop sequence ttcaagaga

reverse complement BACEl-1131 tcgatacctccaatgatca

termination sequence ctttttt

-3'

[00134] An SEC encoding shRNA targeting huntingtin HDl similarly comprises promoter, HDl, a loop sequence, the reverse complement HDl sequence, and a RNA Polymerase III transcription termination sequence. The HDl SECs for use in mice and in humans, respectively, have the following sequences:

SEQ ID No. 66: 5'- cgcgcgtaatacgactcactatagggcgaattgggtacccgctctagaactagtg murine U6 promoter sequence gatccgacgccgccatctctaggcccgcgccggccccctcgcacagacttgtgg gagaagctcggctactcccctgccccggttaatttgcatataatatttcctagtaact atagaggcttaatgtgcgataaaagacagataatctgttctttttaatactagctacat tttacatgataggcttggatttctataagagatacaaatactaaattattattttaaaaa acagcacaaaaggaaactcaccctaactgtaaagtaattgtgtgttttgagactata aatatcccttggagaaaagccttgtttg

enzyme fragment ggccg

HDl tgacagcagtgttgataaa

loop sequence ttcaagaga

reverse complement HDl tttatcaacactgctgtca

termination sequence ctttttt

gaattcctgcagcccgggggatccactagttctagagcggccgccaccgcggtg gagctccagcttttgttccctttagtgagggttaattgcgcg

-3'

SEQ ID No. 75: 5'- human U6 promoter sequence tcgggcaggaagagggcctatttcccatgattccttcatatttgcatatacgataca aggctgttagagagataattagaattaatttgactgtaaacacaaagatattagtaca aaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttt taaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatat atcttgtggaaaggacgaaacacc

HDl tgacagcagtgttgataaa

loop sequence ttcaagaga

reverse complement HDl tttatcaacactgctgtca

termination sequence ctttttt

-3' Accordingly, SECs encoding shRNA targeting huntingtin HD5 have the following sequences:

SEQ ID No. 67: 5'- cgcgcgtaatacgactcactatagggcgaattgggtacccgctctagaactagtg murine U6 promoter sequence gatccgacgccgccatctctaggcccgcgccggccccctcgcacagacttgtgg gagaagctcggctactcccctgccccggttaatttgcatataatatttcctagtaact atagaggcttaatgtgcgataaaagacagataatctgttctttttaatactagctacat tttacatgataggcttggatttctataagagatacaaatactaaattattattttaaaaa acagcacaaaaggaaactcaccctaactgtaaagtaattgtgtgttttgagactata aatatcccttggagaaaagccttgtttg

enzyme fragment ggccg

HD1 ggagtattgtggaacttat

loop sequence ttcaagaga

reverse complement HD1 ataagttccacaatactcc

termination sequence ctttttt

gaattcctgcagcccgggggatccactagttctagagcggccgccaccgcggtg gagctccagcttttgttccctttagtgagggttaattgcgcg

-3'

SEQ ID No. 76: 5'- human U6 promoter sequence tcgggcaggaagagggcctatttcccatgattccttcatatttgcatatacgataca aggctgttagagagataattagaattaatttgactgtaaacacaaagatattagtaca aaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttt taaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatat atcttgtggaaaggacgaaacacc

HD5 ggagtattgtggaacttat

loop sequence ttcaagaga

reverse complement HD5 ataagttccacaatactcc

termination sequence ctttttt

-3'

[00135] SECs encoding shRNA targeting ataxin-1, variant 945 are similarly represented by the following sequences:

SEQ ID No. 68:

cgcgcgtaatacgactcactatagggcgaattgggtacccgctctagaactagtg murine U6 promoter sequence gatccgacgccgccatctctaggcccgcgccggccccctcgcacagacttgtgg gagaagctcggctactcccctgccccggttaatttgcatataatatttcctagtaact atagaggcttaatgtgcgataaaagacagataatctgttctttttaatactagctacat tttacatgataggcttggatttctataagagatacaaatactaaattattattttaaaaa acagcacaaaaggaaactcaccctaactgtaaagtaattgtgtgttttgagactata aatatcccttggagaaaagccttgtttg

enzyme fragment ggccg

ataxin-1 945 aaccaagagcggagcaacgaa

loop sequence ttcaagaga

reverse complement ataxin-1 945 ttcgttgctccgctcttggtt

termination sequence ctttttt

gaattcctgcagcccgggggatccactagttctagagcggccgccaccgcggtg gagctccagcttttgttccctttagtgagggttaattgcgcg

-3'

SEQ ID No. 77:

human U6 promoter sequence tcgggcaggaagagggcctatttcccatgattccttcatatttgcatatacgataca aggctgttagagagataattagaattaatttgactgtaaacacaaagatattagtaca aaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttt taaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatat atcttgtggaaaggacgaaacacc

ataxin-1 945 aaccaagagcggagcaacgaa

loop sequence ttcaagaga

reverse complement ataxin-1 945 ttcgttgctccgctcttggtt

termination sequence ctttttt

-3'

[00136] SECs encoding shRNA targeting alpha- synuclein version 1 and SECs encoding shRNA targeting alpha- synuclein version 2 are represented by the following sequences:

SEQ ID No. 69: 5'- cgcgcgtaatacgactcactatagggcgaattgggtacccgctctagaactagtg murine U6 promoter sequence gatccgacgccgccatctctaggcccgcgccggccccctcgcacagacttgtgg gagaagctcggctactcccctgccccggttaatttgcatataatatttcctagtaact atagaggcttaatgtgcgataaaagacagataatctgttctttttaatactagctacat tttacatgataggcttggatttctataagagatacaaatactaaattattattttaaaaa acagcacaaaaggaaactcaccctaactgtaaagtaattgtgtgttttgagactata aatatcccttggagaaaagccttgtttg

enzyme fragment ggccg

ataxin-1 945 ggaaagacaaaagagggtg

loop sequence ttcaagaga

reverse complement Asynl caccctcttttgtctttcc

termination sequence ctttttt

gaattcctgcagcccgggggatccactagttctagagcggccgccaccgcggtg gagctccagcttttgttccctttagtgagggttaattgcgcg

-3'

SEQ ID No. 78: 5'- human U6 promoter sequence tcgggcaggaagagggcctatttcccatgattccttcatatttgcatatacgataca aggctgttagagagataattagaattaatttgactgtaaacacaaagatattagtaca aaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttt taaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatat atcttgtggaaaggacgaaacacc

ataxin-1 945 ggaaagacaaaagagggtg

loop sequence ttcaagaga

reverse complement Asynl caccctcttttgtctttcc

termination sequence ctttttt

-3'

SEQ ID No. 70: 5'- cgcgcgtaatacgactcactatagggcgaattgggtacccgctctagaactagtg murine U6 promoter sequence gatccgacgccgccatctctaggcccgcgccggccccctcgcacagacttgtgg gagaagctcggctactcccctgccccggttaatttgcatataatatttcctagtaact atagaggcttaatgtgcgataaaagacagataatctgttctttttaatactagctacat tttacatgataggcttggatttctataagagatacaaatactaaattattattttaaaaa acagcacaaaaggaaactcaccctaactgtaaagtaattgtgtgttttgagactata aatatcccttggagaaaagccttgtttg

enzyme fragment ggccg

Asyn2 gggtgttctctatgtaggc

loop sequence ttcaagaga

reverse complement Asyn2 gcctacatagagaacaccc

termination sequence ctttttt

gaattcctgcagcccgggggatccactagttctagagcggccgccaccgcggtg gagctccagcttttgttccctttagtgagggttaattgcgcg

-3' SEQ ID No. 79:

human U6 promoter sequence tcgggcaggaagagggcctatttcccatgattccttcatatttgcatatacgataca aggctgttagagagataattagaattaatttgactgtaaacacaaagatattagtaca aaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttt taaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatat atcttgtggaaaggacgaaacacc

Asyn2 gggtgttctctatgtaggc

loop sequence ttcaagaga

reverse complement Asyn2 gcctacatagagaacaccc

termination sequence ctttttt

-3'

[00137] From these examples and the foregoing descriptions of the possible contents and methods of synthesizing SECs contemplated by the invention, one of ordinary skill in the art would be able to synthesize specific SECs for use in the peptide-polynucleotide compositions and methods of the invention to encode for shRNA having the appropriate target for an identified neurological disease.

[00138] Table 2 shows a list of neurological diseases including the inborn errors of metabolism with neurological involvement, the enzyme deficiency causing each disease, and the animal models associated with each. By associating or conjugating a plasmid DNA molecule that encodes for a missing or deficient enzyme to one of the peptides of the invention, said plasmid DNA molecule may be delivered into the brain of a patient as a treatment for the corresponding disease caused by the enzyme deficiency.

TABLE 2

Typesl/n ilactosaminidase seizures

[00139] By introducing into the cells a sequence of DNA that encodes a shRNA transcript of nucleic acids targeting the genes associated with a neurological or neurodegenerative disease, the expression of the gene is suppressed, and the level of the associated protein in the cells may also be reduced. This suppression and reduction can also be useful in the prevention and treatment of the symptoms of the neurological disease. Examples of known neurological diseases that are treatable with the compositions and methods of the invention are described as follows.

[00140] Alzheimer's disease is a progressive degenerative disorder of the brain characterized by mental deterioration, memory loss, confusion, and disorientation. Alzheimer's disease is characterized by extensive loss of selected neural cell populations accompanied by synaptic injury and astrogliosis. Pathological hallmarks of the disease include the formation of amyloid plaques, neurofibrillary tangles composed of polymerized tau protein and beta amyloid, and neuropil thread formation. Neurodegeneration results from accumulation of amyloid precursor protein, which is processed into beta-amyloid protein by beta amyloid cleaving enzyme 1, including variants thereof. The gene encoding for beta amyloid cleaving enzyme is BACEl. The gene encoding for amyloid precursor protein is APP. Preventing amyloid precursor protein processing into plaque producing forms of amyloid influences has a significant impact on the formation and progression of the disease. Hence, suppressing BACEl expression is of great importance in inhibiting or arresting the disease.

[00141] The neurodegenerative process in Parkinson's disease is also characterized by extensive loss of selected neuronal cell populations accompanied by synaptic injury and astrogliosis. Pathological hallmarks of Parkinson's disease include the formation of Lewy bodies and the loss of dopaminergic neurons in the substantia nigra. Neurodegeneration results from accumulation of a-synuclein protein. Inhibition of the a-synuclein protein produced in neuronal cells is a key to the treatment of Parkinson's disease.

[00142] Huntington's disease is an autosomal dominant neurodegenerative disease that is characterized by involuntary movement, dementia, and behavioral changes. Symptoms include changes in cognitive ability, changes in mood, and changes in coordination and physical movement. These symptoms gradually worsen until the patient dies, approximately 15 to 20 years after the disease's onset. Huntington's disease is caused by a gain of function mutation in the gene encoding for the protein huntingtin (htt). This is the "HD" or "IT- 15" gene, which is located on chromosome 4 at the end of the short arm. Suppression of this gene and reduction in htt in the cells provides effective treatment for the disease.

[00143] Similarly, other neurodegenerative diseases can be effectively treated using similar compositions and methods once the relevant target is identified. One of ordinary skill in the art would be able to tailor the contemplated peptide-polynucleotide compositions of the invention to target any neurological disease using SECs encoding shRNA having the appropriate target. Using the methods of delivery and the compositions of the invention as well as the methods of treatment of the invention, development and progression of the neurodegenerative disease in a patient can be impeded or arrested. This is because the peptide-polynucleotide compositions of the invention, synthesized and purified as disclosed herein, may be effectively delivered according to the methods of the invention into neuronal cells and cell nuclei where the therapeutic polynucleotides can be utilized.

[00144] The invention also includes methods for enhancing siRNA uptake by target cells. In the method, the a peptide-polynucleotide conjugate containing a G domain and a polynucleotide consisting of single- or double-stranded DNA or single- or double-stranded RNA is administered via the carotid or femoral artery of a mammal. Preferably, the polynucleotide is siRNA. Optionally, the DNA is combined with histones in a molar ratio from 1: 1 to 31: 1, as previously described.

[00145] Some of the following Examples illustrate the utility of the peptides of the invention in their ability to associate with DNA and deliver a substance such as an SEC into cells and cell nuclei, whether in vitro or in vivo. The Examples also demonstrate that delivery of the peptides and peptide-polynucleotide compositions and conjugates of the invention may be receptor-mediated and that the peptide-polynucleotide compositions and conjugates are effective in increasing shRNA expression in target cells and in suppressing target gene expression. Examples of preferred embodiments demonstrate that the peptide-polynucleotide compositions and conjugates of the invention are effective in treating neurological and neurodegenerative diseases by suppressing target gene expression. Finally, the Examples demonstrate that the route of delivery of therapeutic polynucleotides affects uptake of the polynucleotide by target cells.

[00146] In general, gene expression can be evaluated by a method to examine RNA levels such as Northern blot analysis, reverse-transcription polymerase chain reaction (RT- PCR), RNAse protection assay, or branched DNA assay. During Northern blot analysis, electrophoresis is used to separate RNA samples separated on agarose gel by size, and then it is detected with a hybridization probe complementary to part of the target sequence. In RT- PCR, the RNA strand is first reverse transcribed into its DNA complement (cDNA) using the enzyme reverse transcriptase, and the resulting cDNA is then amplified using traditional realtime PCR. Neural gene expression can also be evaluated by neural polypeptide levels, including Western blot analysis, immunohistochemistry, or autofluorescence assays. In Western blot analysis, gel electrophoresis is used to separate native or denatured proteins, and then the proteins are transferred to a membrane where they are probed using antibodies specific to the target protein. EXAMPLES:

Example 1

Peptide-DNA conjugate synthesis

[00147] A peptide-DNA conjugate was synthesized via click chemistry reaction between an azide-functional peptide and a 5' alkyne-bifunctional DNA molecule. The conjugate was isolated by precipitation with ethanol. Typical yields using the procedure described below were 80% or greater.

[00148] The peptides were designed to have azide functionality by incorporating a lysine amino acid and reacting its amine with an NHS-PEG-azide molecule (Synthetic Biomolecules). This lysine amino acid derivative was placed between two groups of 3 glycine amino acids, or for the NLS-only peptides, an NLS-(GGG) -PEG- (azide) peptide. The molecular weight of the PEG was either 127 g/mol or 5,000 g/mol, depending on the desired construct. The reaction is described in the following formula:

H

Lysine analog (azide functional) [00149] The forward and reverse primers for the DNA synthesis were synthesized with an alkyne phosphoramidite on the 5' ends by Trilink and the 550 base pair DNA (SEC 1749) was synthesized by PCR using the alkyne functional primers by Vandalia. An alkyne functional phosphoramidite is represented by the following formula:

Alkyne functional phosphoramidite

[00150] The click chemistry reaction between the azide-functional peptide and alkyne functional DNA is shown in Figure 14. The materials used in the reaction are shown in Table 3.

TABLE 3

0.60 603.27 all solutions prepared in 1 :1 t-butanol: water, except DNA which was supplied in water

[00151] The peptide was first prepared along with sodium ascorbate, copper sulfate, and TBTA solutions in 1: 1 tert-butanol water to the concentrations shown in Table 3. For the copper sulfate solution, water was added first to dissolve the copper sulfate and then the tert- butanol was added. For the TBTA solution, sonication was used to achieve complete dissolution. To the tube, the following was added: 104.4 DNA (4.79 mg/mL), 104.4 tert-butanol, 84.4 μL· peptide (5 mg/mL), 10 μΐ_^ sodium ascorbate (28.7 mg/mL), 249,8 μΐ_^ TBTA (3.08 mg/mL), 50.3 μΐ_^ copper sulfate (3.6 mg/mL), and a magnetic stir bar. The materials were magnetically stirred gently for 24 hours at 4°C using the lower molecular weight PEG linker (127 g/mol) and for 48 hours at 4°C using the higher molecular weight PEG linker (5,000 g/mol).

[00152] For precipitation, the magnetic stir bar was removed. To the composition as added 1/10 (water volume) of 3M NaOAc (90.46 μί), 2 times (water volume) 100% ethanol (180.9 μ >. This brought the final volume up to 874.65 μL· The composition was inverted to mix, and white particles were observed. The mixture was stored at -20°C for 2 hours and then centrifuged at 14,000G at 4°C for 15 minutes. Then, the blue/green supernatant was decanted. 1 mL of 70% ethanol was then added and the centrifuging and decanting repeated. Again, 1 mL of 70% ethanol was added and then the centrifuging and decanting repeated. The vial was inverted to allow the DNA pellet to dry at room temperature for 10 minutes. The DNA was brought up in 0.5 mL nuclease-free water and stored at 4°C overnight to allow for complete dissolution. The final DNA yield was 4.34.7 μg in a solution concentration of 869.3 ng^L, providing for an 86.9% yield.

Example 2

GP peptides associate with DNA better than G peptides [00153] The GP peptides of the present invention associate with DNA molecules more efficiently than G peptides. Figure 1 shows an agarose gel in which various peptide- polynucleotide mixtures have been subjected to electrophoresis, after which the gel was stained with ethidium bromide to allow visualization of the DNA. The DNA used in this experiment was linear, double- stranded DNA of approximately 400 base pairs (bp) in size. The G peptide was mixed with the DNA at a peptide to molar ratio DNA of 100: 1, 10: 1, 1: 1, and 0.1: 1. Similarly, the GP peptide was mixed with the DNA at a peptide to DNA molar ratio of 100: 1, 10: 1, 1: 1, and 0.1: 1. If the peptide molecules were associated with the DNA molecules, then the DNA could not run into the gel during electrophoresis. The lanes of the gel labeled "G peptide" show that at a ratio of 100: 1, there is no visible band of DNA at the 400 bp molecular size region of the gel. This indicates that at the 100: 1 ratio, the G peptide is able to associate with the DNA. However, the next lane to the right, labeled "10: 1," shows that a band of DNA is becoming visible at this ratio. This indicates that at the 10: 1 ratio, the G peptide is not able to associate sufficiently with the DNA to keep all the DNA from running into the gel in response to the electrophoresis. In contrast, the lanes of the gel labeled "GP peptide" show that at both the 100:1 ratio and the 10: 1 ratio, the GP peptide associates sufficiently with the DNA to keep all detectable DNA from running into the gel in response to the electrophoresis. Thus, the GP peptide of the invention is about tenfold more effective at associating with DNA than the G peptide is alone.

[00154] The characteristic that the GP peptide of the invention is about tenfold more effective at associating with DNA than the G peptide is specific to DNA; it does not pertain to siRNA. This result is predictable, in light of the present invention, because the protamine (P) domain is included in peptides of the present invention specifically because it provides an amino acid sequence that associates with and compacts DNA. It was not expected that the P domain of a GP peptide would provide any additional association capability to siRNA beyond that provided by the G domain alone.

[00155] Figure 2 shows an agarose gel in which various peptide-siRNA mixtures have been subjected to electrophoresis, after which the gel was stained with ethidium brominde to allow visualization of the siRNA. The siRNA used in this experiment was linear, double- stranded RNA of approximately 19 base pairs in size. The G peptide was mixed with this siRNA at peptide to siRNA molar ratios of 100: 1, 10: 1, 1: 1, and 0.1: 1. Similarly, the GP peptide was mixed with this siRNA at peptide to siRNA molar ratios of 100: 1, 10: 1, 1: 1, and 0.1: 1. If the peptide molecules were associated with the siRNA molecules, then the siRNA could not run into the gel during electrophoresis. If the siRNA does not run into the gel in response to electrophoresis, it becomes visible as a bright, fuzzy band at the bottom of the gel. Comparison of the lanes of the gel labeled "G peptide" and "GP peptide" shows that the G and GP peptides are essentially equivalent in terms of the ratios at which the peptides sufficiently associate with the siRNA to keep the siRNA from running into the gel in response to electrophoresis. Taken together, Figures 1 and 2 show that although both peptides are able to associate with siRNA molecules, the GP peptide of the invention is uniquely able to associate with DNA molecules.

Example 3

GP and GPN peptides associate with DNA [00156] The GPN peptides of the invention associate with DNA, including plasmid

DNA. The plasmid used in this example is circular plasmid DNA p_eGFP-cl (SEQ ID No. 37), GenBank Accession No. U55763. The G peptides, GP peptides, or GPN peptides of the invention were mixed in a test tube with circular plasmid DNA p_eGFP-cl in molar ratios of 100: 1, 500: 1, and 1000: 1 molecules of peptide per molecule of DNA. The mixtures were each then subjected to gel electrophoresis using standard techniques known in the art. Figure 3 is a photograph of the resulting gel, after staining with ethidium bromide to allow for visualization of the plasmid DNA. The visible presence of the DNA in the lanes of gel for the 100: 1, 500: 1, and 1000: 1 ratios of G peptides to plasmid DNA indicates that the G peptides cannot efficiently associate with the DNA and prevent it from running into the gel during electrophoresis. In contrast, the absence of much DNA in the lanes of gel for the 1000: 1 ratio of GP peptides to plasmid and the presence of a visible amount of DNA at the top of the gel trapped near the loading well indicates that GP peptides are able to associate with plasmid DNA. The lanes for GPN peptide to plasmid mixtures show that, at the ratios of 500: 1 and 1000: 1, the GPN peptides of the invention are able to associate with plasmid DNA.

Example 4

GPN peptides transfect Neuro2a cells better than 293 cells, indicating that the uptake of GPN-plasmid into Neuro2a cells is receptor-mediated

[00157] The GPN peptides of the invention serve to transfect cells that express the appropriate cell-surface receptors utilized by the rabies virus glycoprotein domain better than they transfect cells that are known not to express such receptors. Figure 4 contains photographs showing representative results of in vitro cell transfection experiments using HEK 293 cells, which are a cell line known not to express nicotinic acetylcholine receptors (nAchRs), and using Neuro2a ("N2A") cells, which are a cell line known to express nAchRs (Kumar et al., 2007). Cells in various conditions in the experiment were exposed to an equal amount, 2.0 μg, of plasmid DNA encoding an enhanced green fluorescent protein p_eGFP- cl, which is labeled in Figure 4 as p_eGFP (SEQ ID No. 37). The plasmid DNA was mixed with G, GP, or GPN peptides of the present invention at a molar ratio of 1000: 1 molecules of peptide per plasmid molecule. For the Neuro2a cells, the plasmid DNA was also mixed with GPN peptides of the invention at a molar ratio of 1250: 1. The cells were incubated at 37°C for 48 hours and then examined by fluorescence microscopy for evidence of green fluorescent protein (GFP) expression, which would indicate successful, functional transfection of the cells by the plasmid DNA, because the plasmid would have been transported to the nucleus of the cell and utilized there, resulting in the production of GFP in the cell.

[00158] The upper row of photographs shows a paucity of functionally transfected cells in those conditions in which the cells were HEK 293 cells, which lack receptors for receptor- mediated uptake of the G, GP, and GPN peptides. The lower row of photographs shows a similar paucity of functionally transfected cells where the cells were Neuro2a cells, which have receptors for the rabies virus glycoprotein, and where the peptide applied was the G or GP peptide of the invention. In contrast, the two rightmost photographs in the lower row show numerous functionally transfected cells in Neuro2a cells where the plasmid was used with the GPN peptide of the invention. Moreover, the number of functionally transfected Neuro2a cells is increased where the 1250: 1 ratio of GPN peptide was used in vitro compared to the 1000: 1 ratio of GPN peptide to plasmid DNA. These results indicates that the nuclear localization signal domain included in the GPN peptide of the invention provides greater functional transfection of cells with DNA. This is consistent with the interpretation that the NLS domain of the peptides of the invention does cause improved successful transport of DNA into the nucleus of a cell, where it can be utilized. The results also show a dose effect on the number of functionally transfected cells.

Example 5

GPN peptides transfect linear DNA (SECs) into Neuro2a cells and result in functional shRNA expression as evidenced by target gene suppression

[00159] In another experiment, the GPN peptides of the invention were mixed with

DNA comprising Silencer Expression Cassettes (SECs) encoding short, hairpin RNA

(shRNA) targeting beta amyloid cleaving enzyme type 1 (BACE1) mRNA. These SECs were comprised of the DNA sequence disclosed in SEQ ID. No. 11. The GPN peptides were mixed with the SECs in molar ratios of 30: 1, 50: 1, 70:1, 80: 1, 90: 1, 100:1, 110: 1, and 120: 1 of GPN peptide molecules of the invention to SEC molecule. The GPN-SEC compositions were then used to transfect the SECs into Neuro2a cells, which are of a murine neuroblastoma cell line. Neuro2a cells are known to express BACE1, the growth factor receptor (p75^ntr), and the nicotinic acetylcholine receptor (nAchR) that is a receptor for the rabies virus glycoprotein. Two days after transfection of the cells, total RNA was harvested from the cells using standard techniques, and a reverse-transcription polymerase chain reaction (RT-PCR) assay was used to measure the amount of BACE1 mRNA in the cells.

The reverse transcription step of the RT-PCR assay was performed using the Stratagene

ProSTAR First Strand Reverse Transcription kit, Cat# 200420, [Agilent Technologies, Santa

Clara, California] following the manufacturer's instructions, and using the oligo dT primers provided with the kit. The Polymerase Chain Reaction step of the RT-PCR assay was performed using the BACEl Assay on Demand from Applied Biosystems, Inc., (Carlsbad, California), Cat# Mm00478664-ml using TaqMan Universal PCR Mix with the PCR reaction started with a 15 min hold at 95 degrees Centigrade, then 45 cycles consisting of 15 seconds at 95 degrees C, 30 seconds at 50 degrees C, and 30 seconds at 72 degrees C. The amount of BACEl mRNA was normalized to the amount of Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in the same cells (assayed in a parallel RT-PCR reaction) to control for any differences in the number of cells harvested and the amount of starting RNA utilized. Finally, the ratio of BACEl mRNA to GAPDH mRNA in cells that had been "mock transfected," i.e., had not received any SECs, was set to 100%, and the ratio of BACEl mRNA to GAPDH mRNA in treated cells was set relative to the mock transfected cells.

[00160] The graph in Figure 5 shows that, compared to the mock transfected cells,

Neuro2a cells transfected with SECs encoding anti-BACEl shRNA using the GPN peptides of the invention at a 50: 1 ratio of GPN to SEC have only about 50% as much BACEl mRNA. This indicates that about 50% BACEl suppression has been achieved. Similar suppression was obtained using a GPN to SEC ratio of 30: 1, and measurable suppression was also obtained using other GPN to SEC ratios. These results show that the peptides of the invention are capable of delivering functional SECs into cells. The SECs delivered into the cells using the peptides of the invention are used by the cell to process shRNA transcripts that are effective at suppressing the expression of mRNA of the target genes, as expected.

Example 6

PNG peptides yield greater uptake and expression of SEC in vivo than GNP peptides; PN- PEG(2k)-G can also provide high levels of expression

[00161] The PNG peptides of the invention are composed of the DNA condensing protamine domain on the amino terminal end of the peptide, followed by the nuclear localization signal domain, and followed by the rabies virus glycoprotein domain on the carboxyl terminal end of the peptide. PNG peptides result in greater uptake and expression of DNA comprising a Silencer Expression Cassette (SEC) in the mammalian brain upon in vivo intracranial infusion than GNP peptides. GNP peptides of the invention are composed of the rabies virus glycoprotein domain on the amino end of the peptide, followed by the nuclear localization signal domain, and followed by the DNA condensing protamine domain on the carboxyl end of the peptide.

[00162] Figure 6 shows the amount of shRNA transcripts from SECs compared to the amount of mRNA for Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) that is measured in the brain tissue of mice. The brain tissue of the mice had been directly injected by intracranial infusion with a constant amount, 10 μg, of SECs combined with various peptides of the invention or other transfection reagents. The ratio of shRNA to GAPDH is shown on the y-axis in Figure 6 on a logarithmic scale, where the value for an untreated animal has been arbitrarily set to one. As shown in Figure 6, in four mice receiving PNG peptides combined with an SEC at a ratio of 50: 1 molecules of PNG peptide to DNA, the expression of shRNA transcripts in the injected hemisphere of the brain was 26.08 to 704.95 (average: 204.1 times greater than that in a non-treated mouse. In contrast, in four mice receiving GNP peptides combined with an SEC at a ratio of 50: 1 molecules of GNP peptide to DNA, the expression of shRNA transcripts in the injected hemisphere in the brain was only 2.47 to 18.31 (average: 6.5) times greater than that in a non-treated mouse. These results show that the organization of the domains of the peptide in the PNG peptide resulted in an improvement in the average amount of shRNA transcripts resulting in the brain of 31.3 times, compared to the amount obtained using the GNP peptide.

[00163] The same experiment was also conducted using the PN-PEG(2k)-G peptides of the invention mixed with SECs. Figure 6 shows that PN-PEG(2k)-G peptides bound to SECs were capable of functional delivery of the DNA into brain cells, resulting in shRNA transcription. In one mouse, the resulting shRNA transcription level was 76.11 times greater than in an untreated mouse. These results indicate that PN-PEG(2k) peptides can also provide high levels of expression of SEC in the brain.

Example 7

PNG peptides deliver linear DNA (SECs) into cells, where it co-localizes with cell nuclei, when injected into the brain

[00164] To further confirm the transfection of SECs into cells upon delivery of the

DNA into the mammalian brain by direct intracranial injection, DNA that had been labeled with AlexaFluor 750 was mixed with PNG peptides of the invention and injected into the striatum of a mouse. AlexaFluor 750 is a fluorophore that fluoresces red upon appropriate illumination under fluorescence microscopy. Twenty-four hours later, the mouse was euthanized, transcardially perfused, and the brain was harvested. Following sectioning through the striatum, the tissue was stained with Dapi, a stain which makes cell nuclei visible under fluorescent microscopy.

[00165] Figure 7 shows the appearance of the tissue under fluorescent microscopy under illumination for AlexaFluor 750 and under illumination for Dapi. The images in Figure 7 reveal the DNA in the striatum by virtue of the AlexaFluor 750 labeling of the DNA. Overlap of the AlezaFluor 750 image with the Dapi image of the same tissue section indicates that the DNA and the cell nuclei are co-localized, which is consistent with cellular uptake and nuclear localization of the DNA following delivery using the PNG peptides of the invention. The bottom image of Figure 7 also shows labeled DNA and Dapi co-localization in cells of the corpus callosum of the mouse.

[00166] The ability of the PNG peptides of the invention to deliver DNA to the nucleus of the cells, as shown in Figure 7, may be contrasted with the localization of DNA in cell lysosomes or endosomes, to the relative exclusion of the cell nucleus, as seen in Figure 5 of Liu et al., 2009.

Example 8

PN-PEG(2k)-G peptides transfect linear DNA (SECs) across the blood-brain barrier and into brain cells in vivo when delivered via the carotid artery

[00167] The PN-PEG(2k)-G peptide of the invention, when mixed with linear double- stranded comprising Silencer Expression Cassettes (SECs) and infused via the carotid artery of a mammal, are capable of delivering the SECs across the blood-brain barrier and into the brain. This is evidenced by the presence of shRNA transcripts from the SECs in the brain. Figure 8 shows the results of the analysis of brain tissue from the left and right hemispheres of mice brains in various experimental treatment groups. Mice were pre-catheterized with a catheter positioned in the left internal carotid artery with the catheter oriented towards the head of the animal (Charles River Labs.), and the proximal end of the catheter was accessed and used to deliver 300 μΐ_^ of fluid to the carotid artery at a rate of 600 μί/ιηίη, i.e., an infusion lasting 30 seconds.

[00168] In the saline group (n = 3 mice), only saline was infused. In the SECs group

(n = 7 mice), 150 μg SECs in phosphate buffered saline solution was infused. In the PNG50: 1 group (n = 5 mice), PNG peptides of the invention were mixed with SECs at a ratio of 50: 1 molecules of peptide to DNA in phosphate buffered saline solution, and 300 μΐ_^ containing 150 μg SEC (and the PNG peptide) was infused into each mouse via the carotid artery. In the PN-PEG(2k)-G group (n = 5 mice), PN-PEG(2k)-G peptides of the invention were mixed with SECs at a ratio of 50: 1 molecules of peptide to DNA in phosphate buffered saline solution, and 300 μΐ_^ containing 150 μg SEC (and the PN-PEG-G peptide) was infused into each mouse via the carotid artery. Twelve days later, the mice were euthanized and transcardially perfused with saline, only. The brain of the mouse was quickly removed and divided into the left and right hemispheres, and the hippocampal region of each hemisphere was dissected away from the remaining brain tissue and frozen on dry ice. Each sample of frozen tissue was later homogenized. Total RNA was isolated from the sample and used to assay for shRNA transcripts, which were normalized to the amount of small nuclear RNA (SNO RNA) in the same sample by reverse transcription polymerase chain reaction (RT- PCR). Specifically, total RNA was isolated from mouse brain tissue samples using the mirVana miRNA isolation kit (Applied Biosystems, Foster City, CA) following the manufacturer's recommended protocol. The tissue was homogenized in lysis/binding buffer using an Omni-Tip homogenization probe (Omni International, Kennesaw, GA). The total RNA was treated with DNase using the Turbo-DNA-free kit (Applied Biosystems, Foster City, CA). cDNA was generated from 10 ng of total RNA using shRN A- specific reverse transcription primers and the Taqman® MicroRNA reverse transcription kit (Applied Biosystems, Foster City, CA). Custom Taqman® small RNA assays were designed for the MB 1749 shRNA by software provided by Applied Biosystems (Applied Biosystems, Foster City, CA). In these expression studies the endogenous control snRNA known as SNO202a was used to normalize the expression of the shRNAs across samples. Real-time PCR reactions were set up using the CAS 1200 Precision Liquid Handling System (Corbett Life Science, San Francisco CA) and the generated cDNA. Real-time PCR was performed using the Applied Biosystems 7900HT Fast real-time PCR system. The average of the amounts of SNO202a snRNA in each sample from the left hemispheres of mice in the saline control group was arbitrarily assigned the value "1" (left-most bar in Figure 8).

[00169] Figure 8 shows the amount of shRNA transcripts, normalized to SNO RNA in the other treatment groups, as compared to the saline group, by hemisphere. In mice in the PN-PEG(2k)-G group, a statistically significant (p < 0.01), 5.5-fold increase in shRNA transcript quantity over that in the saline control group was found in the samples of the left hemisphere, ipsilateral to the carotid artery receiving the infusion. This increase is also significantly greater than the amount in the left hemispheres of the mice in the SECs group (p < 0.01). Similarly, in the PN-PEG(2k)-G group, a statistically significant (p < 0.05), fourfold increase in the shRNA transcript quantity over that in the saline control group was found in the samples of the right hemisphere, contralateral to the carotid artery receiving the infusion. This increase is also significantly greater than the amount in the right hemispheres of mice in the SECs group (p < 0.05). These results indicate that the PN-PEG(2k)-G peptides of the invention are capable of delivering functional, linear DNA comprising Silencer Expression Cassettes into the brain from an infusion site in the carotid artery.

[00170] For unknown reasons, contrary to the results shown in Example 7 and Figure

7, in which both PNG and PN-PEG(2k)-G peptides were effective at delivering SECs into brain cells upon direct intracranial infusion, the experiment of Figure 8 shows that mice receiving SECs mixed with the PNG peptide did not exhibit an increase in shRNA transcripts in the brain.

[00171] The results of Examples 5, 6, and 7 together indicate that the order of the domains of the peptide chain (P, N, and G), the inclusion of the nuclear localization signal domain generally, and the separation of the G domain from the other domains, i.e., by PEG(2k), are useful features of the PN-PEG(2k)-G peptide of the invention for the purposes of achieving successful delivery of SECs to the brain from a method of delivery using the carotid artery.

Example 9a

GN peptides can be directly conjugated to linear DNA (SECs) using "click chemistry" [00172] The GN peptides of the invention can be covalently attached to the 5' end of a strand of DNA using a Huisgen 1,3-dipolar cycloaddition reaction, which is a chemical reaction between an azide and an alkyne that is catalyzed by copper(I) (Cu¹), known as a "click chemistry" reaction. Optionally, a GN peptide can be covalently attached to either of the 5' ends of a linear, double- stranded DNA molecule. Optionally, a GN peptide can be covalently attached to the 5' end of one DNA strand of a linear, double- stranded DNA molecule, and another GN peptide can be covalently attached to the 5' end of the complementary DNA strand of that molecule.

[00173] A method of making the GN-SEC conjugate of the invention consists of conjugating the GN peptide of the invention to the 5' end of a double-stranded DNA molecule by a "click chemistry" reaction. The GN peptide is modified to have a lysine amino acid residue located between the G domain and the N domain. The lysine amino acid residue is modified with NHS-(PEO)₄-azide to have an azide on the side chain. Alternatively, the modified lysine amino acid residue could be located at either the amino end of the G domain or at the carboxyl end of the N domain. The 5 Of a strand of the DNA molecule to which the GN peptide is to be conjugated is modified to have an alkyne group. For example, a double- stranded DNA molecule with an alkyne modification at the 5' end of one or both strands can be generated by synthesizing primers by Polymerase Chain Reaction (PCR) using an alkyne functional phosphoramidite, such as 5'-hexynyl phosphoramidite and then using those primers in the PCR synthesis of the double-stranded DNA molecule, using well-known PCR methods in the art.

[00174] A non-limiting example of the method of conjugating and purifying the GN peptide to DNA to make the GN-SEC conjugates of the invention is as follows. The GN peptide is modified as described above to have an azide functional group. A double- stranded DNA molecule having 530 base pairs is modified to have an alkyne group on the 5' end of both of its strands. The 5 '-alkyne functional DNA (344,798 g/mol; Vandalia) is prepared at a concentration of 2.9 mg/mL in nuclease-free water (NF water). An aliquot of 0.5 mg of the azide functional GN peptide (4875.29 g/mol; Abgent) is dissolved in 500 microliters of NF water. To 172.4 μϊ_^ of DNA solution, the following is added: 14 μL· of peptide solution, 10 μΐ_^ of sodium ascorbate solution (28.7 mg/mL NF water), 145 μΐ_^ of TBTA (Tris-[(l-benzyl- lH-l,2,3-triazol-4-yl)methyl]amine, also known as tris-(benzyltriazolylmethyl)amine) (0.053 mg/mL), and 10 μΐ_^ of copper sulfate 5H₂0 solution (14.48 mg/mL NF water). TBTA is a stabilizing ligand for Cu¹. The molar ratio for all components of the reaction is 2 peptide : 1000 sodium ascorbate : 10 TBTA : 400 copper sulfate to 1 DNA molecule. The reaction proceeds spontaneously. The reaction is allowed to run to completion by gently magnetically stirring the solution for one hour at room temperature and then for 18 hours at 4°C.

[00175] The concentration of the peptide-DNA conjugate in the completed reaction is

1.4 μg/μL. The solution is diluted with 0.9% sterile saline to a concentration of approximately 1 μg/μL. This diluted solution is run through a NAP™-5 purification column with 0.9% saline as the elution buffer to remove small molecules, such as catalysts and ligands. The resulting solution may be used to transfect cells in vitro, or it may be administered in vivo to mammals by arterial delivery or direct intracranial infusion. Analysis of this solution was performed by high pressure liquid chromatography (HPLC) using a negatively charged cation exchange column (CEX) and standard methods known to those skilled in the art.

[00176] The results of the HPLC analysis are shown in Figure 9. The top graph in

Figure 9 shows the HPLC analysis of the DNA alone. The bottom graph shows the peptide- DNA mixture (at a 2: 1 molar ratio) when the copper catalyst for the "click chemistry" reaction has not been added. These two graphs are similar, which indicates, as expected, that in the absence of the copper catalyst, the peptide and DNA are not conjugated. In contrast, the middle graph of Figure 9 shows the HPLC analysis of the peptide-DNA mixture following the "click chemistry" reaction that was induced by inclusion of the copper catalyst in the reaction mixture, as previously described. This HPLC tracing is clearly different from that of the other two tracings, and it shows a broadening of the peak due to prolonged interaction of the negatively charged column with DNA conjugated to positively charged peptides. This indicates that the formation of peptide-DNA conjugates has occurred, as expected.

Example 9b

A preferred method for synthesizing GN-SEC conjugates [00177] In the method described in Example 9a for conjugating the GN peptide of the invention to polynucleotides, the inclusion of TBTA in the reaction mixture is preferable not only because it stabilizes the copper catalyst, but also because it prevents the copper in the reaction mixture from degrading the DNA such that the resulting reaction product is not the desired product. A preferred method for synthesizing GN-SEC conjugates of the invention includes TBTA in the reaction mixture in a molar excess of 2: 1 compared to the copper sulfate. The TBTA is also completely dissolved in a 1: 1 solution of water and tert-butanol to maintain TBTA in solution. A molar excess of peptide versus DNA is used to help the conjugation reaction proceed to completion. The molar ratio for all components of the reaction is 30 peptide : 1000 sodium ascorbate : 1000 TBTA : 500 copper sulfate : 1 DNA molecule.

[00178] As a non-limiting example, the GN peptide molecule is modified with an azide group on the side chain of a lysine between the G domain and the N domain, and it is conjugated by "click chemistry" reaction to the 5' ends of a double- stranded DNA molecule having 530 base pairs and an alkyne functional group on the 5' ends of both strands. The method for conjugating the peptide to DNA in this example is as follows. 91.1 μΐ_^ of tert- butanol is added to an equal volume (91.1 μί) of 5 '-alkyne functional DNA (344,798 g/mol; Vandalia) (5.49 mg/mL). The solution is swirled gently until the DNA is completely dissolved. The azide functional GN peptide (4875.29 g/mol; Abgent) is dissolved in a 1: 1 mixture of water and tert-butanol to a final concentration of 5 mg/mL, and 42.4 μϊ_^ of this solution is added to the DNA solution. Then, the following is added: 10.0 μΐ_^ sodium ascorbate (28.7 mg/mL in 1: 1 water to tert-butanol), 249.8 μΐ. TBTA (3.08 mg/mL in 1: 1 water to tert-butanol), and 50.3 μΐ_^ copper sulfate (3.6 mg/mL in 1: 1 water to tert-butanol). The solution is gently magnetically stirred at 4°C for 18 hours. The catalysts and excess peptide can be removed using the methods described in examples 9 or 10. Also as described in examples 9 and 10, the solvent can be changed from 1: 1 water to tert-butnaol to phosphate buffered saline in preparation for cell transfection in vitro, or for administration in vivo to mammals by arterial delivery or direct intracranial infusion.

[00179] The GN-SEC peptide-polynucleotide conjugates made as described in this example can be analyzed and purified by subjecting the GN-SECs to electrophoresis in an agarose gel, using methods known to those skilled in the art of DNA electrophoresis.

Electrophoresis of GN-SEC conjugates in a 2% agarose gel containing ethidium bromide allows visualization of the DNA under ultraviolet light, as is shown in Figure 10. The leftmost lane and the rightmost lane of the gel in Figure 10 contain a standard DNA molecular weight ladder. The lane second from the left ("lane two") shows DNA comprising

SECs encoding for shRNA targeting BACE1 (SEC1749, as in SEQ ID No. 30) prior to conjugation to a GN peptide. The lane third from the left ("lane three") shows the GN-SEC conjugates resulting from a "click chemistry" reaction as previously described. As expected, the band of DNA in the lane three is shifted higher on the gel, which indicates a slower migration and, therefore, slightly more positive charge of the GN-SEC molecules as compared to the DNA band in lane two that consists of unconjugated SECs. The results of the gel electrophoresis thus provide a visual confirmation of the formation of the GN-SEC conjugates by "click chemistry" reaction.

Example 10

A method for purifying GN-SEC conjugates [00180] When excess peptide is used in the "click chemistry" reaction, a method other than the NAP™-5 size exclusion column described in Example 9a is needed for purification of the conjugates, because the unreacted peptide may be large enough to pass through the column along with the conjugated DNA. The present invention includes the following method of purification of the peptide-polynucleotide conjugates. Spin columns with sufficiently high molecular weight cut-offs (MWCO) can be used to remove catalysts and peptides and also to exchange the solvent for saline or phosphate buffered saline (PBS). The general method is as follows.

[00181] 250 μΐ_^ of water is added to the reaction mixture, and the mixture is placed on ice for 20 minutes to precipitate the TBTA. The tube is centrifuged at 10,000G for 3 minutes at 4°C. The supernatant is collected, and 400 μΐ_^ is transferred to a Microcon® YM-10 regenerated cellulose 30,000 MWCO spin column that has been preconditioned with PBS. The spin column is then centrifuged at 14,000G at 4°C until approximately 100 remains in the column, approximately 20 minutes. Additional reaction solution can be added and passed through the column, decreasing the volume and concentration of the conjugate. The solvent can be exchanged by adding the desired new solvent, i.e., saline or PBS, and passing the solution through the column, which is centrifuged as before. Repeating this procedure several times results in a concentrated and purified conjugate solution in a physiological buffer. Typical conjugate yields with this method are 20%.

Example 11

A preferred method for purifying GN-SEC conjugates [00182] While purification of GN-SEC conjugates of the invention using the spin column method described in Example 10 is feasible, it is laborious to perform when a large quantity of GN-SEC conjugates is desired. The present invention also includes the following method for purification of the peptide-polynucleotide conjugates of the invention. This method provides at least a 70% yield of peptide-polynucleotide conjugates, which is much higher than the yield achieved with the spin column method.

[00183] In Example 9b, the completed "click chemistry" reaction mixture consists of the desired peptide-polynucleotide conjugates, residual unreacted free peptides, residual unreacted free DNA, copper sulfate, sodium ascorbate, TBTA, nuclease-free water (NF water) and tert-butanol. To this mixture, 0.1 volume (relative to the NF water in the solution) of 3 Molar sodium acetate is added. Next, 2 volumes (relative to the NF water) of ice-cold 100% ethanol is added. Small, white precipitate is typically observed. The resulting mixture is then incubated at -20°C for 1 hour. After incubation, the mixture is centrifuged at 14,000G for 15 minutes at 4°C. The supernatant is then discarded and replaced with 70% ethanol. This mixture is also centrifuged at 14,000G for 15 minutes at 4°C. The supernatant is again discarded, and the pellets of peptide-polynucleotide conjugates are allowed to dry at room temperature for about 10 minutes. Finally, the pellets are re-suspended in phosphate buffered saline. Recovery of the peptide-polynucleotide conjugates is typically about 70-95% of the theoretically expected amount, which is based upon starting quantities of DNA. The recovery yield can achieve up to 98% of the theoretically expected amount using this method. [00184] This purification method is successful due to (1) the optimum salt concentrations resulting from adding 3 Molar sodium acetate, at 1/10 of the starting volume of NF water, to the starting volume of the mixture, (2) the optimum alcohol volume of 2 times the starting volume of NF water, and the optimum precipitation time of 1 hour at -20°C. Using other volumes or other times can also result in successful recovery of the peptide- polynucleotide conjugates, but at suboptimum yields.

Example 12

Enhanced expression of shRNA in Neuro2a cells when transfected by GN-SEC conjugates [00185] In the conjugated form, the GN peptide portion of the GN-SEC conjugates continues to facilitate entry into cells and/or nuclear localization of DNA in cells in vitro, and the DNA portion of the conjugates also continues to be functional, such that shRNA transcripts are produced by the cell utilizing the encoded Silencer Expression Cassettes.

[00186] Figure 11 shows the results of an assay for shRNA transcripts from DNA comprising a Silencer Expression Cassette encoding shRNA targeting BACE1, i.e., SEC1749 (SEQ ID No. 30). In the experiment reflected in Figure 11, Neuro2a cells had been transfected in a six-well cell culture plate 48 hours earlier either with 2 μg GN-SEC conjugates (conjugate number 14453-26, where the SEC is SEC1749), or with 2 μg SECs using the commercial transfection reagent Transit-Neural. The results demonstrate that the GN-SEC conjugate can be transcribed as efficiently as unmodified SECs, which indicates that the covalently bound peptide does not interfere with transcription. The results show that the GN-SEC can be transcribed as efficiently as unmodified SEC, indicating that the covalently bound peptide does not interfere with transcription. The GN-SEC conjugates used in this experiment were synthesized as described in example b and purified as described in example 10.

[00187] Figure 11(a) shows the results of an assay for shRNA transcripts from DNA comprising a Silencer Expression Cassette encoding shRNA targeting BACE1, i.e., SEC1749 (SEQ ID No. 30), in cell lysates from Neuro2a cells. The cells had been transfected in a six- well cell culture plate 48 hours earlier with 10 μg GN-SEC conjugates. No additional cell transfection reagents were used in the experiment shown in Figure 12. The results show a 2.1-fold increase in the amount of shRNA transcripts in the Neuro2a cells obtained using the GN-SEC conjugates than was obtained using the SECs alone. This indicates that the GN peptide portion of the conjugate enhances functional transfection of the cells. An assay for shRNA transcripts from DNA comprising a Silencer Expression Cassette encoding SEC1749 is hereinafter referred to as the "1749 assay" or the "1749 shRNA assay."

[00188] Another GN-SEC conjugate (number 14453-64) were made according to the method described in Example 9b and purified according to the method described Example 10. Figure 11(b) shows the results of the 1749 shRNA assay for cells treated with SECs and GN- SEC conjugates transfected with the commercial transfection reagent Transit-Neural. The results show that the GN-SEC conjugate can be transcribed as efficiently as unmodified SECs, which indicates that the covalently bound peptide does not interfere with transcription. Figure 11(c) shows the results of the 1749 shRNA assay for cells treated with SECs and GN- SEC conjugates transfected without an additional transfection reagent. The results show a 2.5 to 3.5-fold increase in the amount of shRNA transcripts in the Neuro2a cells obtained using GN-SEC conjugates than was obtained using the SECs alone. This indicates that the GN peptide portion of the GN-SEC conjugates enhances the functional transfection of the cells.

[00189] Another GN-SEC conjugate (14453-92) was made according to the method described in Example 9b and purified according to the method described Example 11. Figure 11(d) shows the results of the 1749 shRNA assay for cells treated with SECs and GN-SEC conjugates transfected with the commercial transfection reagent Transit-Neural. The results show that the GN-SEC conjugates can be transcribed as efficiently as unmodified SECs, which indicates that the covalently bound peptide does not interfere with transcription. Figure 11(e) shows the results of the 1749 shRNA assay for cells treated with SECs and the GN-SEC conjugates transfected without an additional transfection reagent. The results show a 2.1 -fold increase in the amount of shRNA transcripts in the Neuro2a cells obtained using GN-SEC conjugates than was obtained using SECs alone. This indicates that the GN peptide portion of the conjugate enhances functional transfection of the cells. [00190] Another GN-SEC conjugate (14596-63) was made according to the method described in Example 9b and purified according to the method described Example 11. Figure 11(f) shows the results of the 1749 shRNA assay for cells treated with SECs and the GN-SEC conjugates transfected with the commercial transfection reagent transit-Neural. The results show that the GN-SEC can be transcribed as efficiently as unmodified SECs, indicating that the covalently bound peptide does not interfere with transcription. Figure 11(g) shows the results of the 1749 shRNA assay for cells treated with SECs and the GN-SEC conjugates without an additional transfection reagent. The results show a 1.9-fold increase in the amount of shRNA transcripts in the Neuro2a cells obtained using the GN-SEC conjugate than was obtained using SECs alone. This indicates that the GN peptide portion of the conjugate enhances functional transfection of the cells.

Example 13

GN-SEC conjugates transfect linear DNA (SECs) across the blood-brain barrier and into brain cells in vivo when delivered via the carotid artery

[00191] The GN-SEC conjugates of the example, when infused via the carotid artery of a mammal, are capable of delivering the SECs across the blood-brain barrier and into the brain, which is evidenced by the presence of shRNA transcripts from the SECs in the brain.

Figure 12 shows the results of one additional experimental group included in the experiment described in Example 8. The additional experimental group (n = 5 mice) received an infusion of 300 μΐ_^ of solution containing GN-SEC conjugates of the invention in an amount equivalent to 150 μg of DNA. The infusion was performed at a rate of 600 μΕ/ηιίη into a catheter previously positioned into the left carotid artery of the mouse. As with the other mice in this experiment, the mice were euthanized twelve days later and transcardially perfused with only saline. The brain of the mouse was quickly removed and divided into the left and right hemispheres, and the hippocampal region of each hemisphere was dissected away from the remaining brain tissue and frozen on dry ice. The relative amounts of shRNA transcripts in the tissue samples were determined using the RT-PCR assay, as previously described in Example 8.

[00192] Figure 12 contains the same data as Figure 8 for the saline, SECs, PNG 50: 1, and PN-PEG(2k)-G mice groups. In addition, Figure 12 shows the results for the group receiving GN-SEC conjugates. The white bar that is fifth from the left of Figure 12 shows the results from the left hemisphere of the five mice in this group, and the white bar at the far right shows the results from the right hemisphere of the five mice in this group. The figure shows that the brain samples of the left and right hemispheres of the mice treated with GN- SEC conjugates contained 4.2 and 4.3 times more shRNA transcripts relative to SNO RNA by the RT-PCR assay compared to mice receiving saline only. This observed difference is statistically significant (p < 0.05). These results indicate that the GN-SEC conjugates of the invention are capable of delivering functional DNA comprising a Silencer Expression Cassette to the brain of a mammal upon administration via the carotid artery of the mammal.

Example 14

NG-SEC conjugates transfect linear DNA (SECs) across the blood-brain barrier and into brain cells in vivo when delivered via the carotid artery

[00193] The NG-SEC conjugates of the invention are also capable of delivering SECs across the blood-brain barrier and into the brain, which is evidenced by the presence of shRNA transcripts from SECs in the brain following infusion of the NG-SEC conjugates via the carotid artery of a mammal.

[00194] NG-SEC conjugates are formed by conjugating an NG peptide to a DNA molecule comprising a Silencer Expression Cassette. For the purposes of this example, the NG-SEC conjugates have a covalent bond between the peptide and double-stranded DNA located at a position between the carboxyl end of the N domain and the amino end of the G domain of the peptide. The covalent bond chemically attaches the peptide to the 5' end of both strands of DNA. [00195] Similar to the experiment of Example 8, in the example reflected in Figure 13, mice were pre-catheterized with a catheter positioned in the left internal carotid artery, oriented towards the head of the animal (Charles River Labs.), with the proximal end of the catheter externalized for later access. Several days later, 300 μΐ_^ of fluid was delivered via the proximal end of the catheter into the carotid artery at the rate of 600 μΕ/ηιίη, i.e., an infusion lasting 30 seconds. In the SEC group (n = 4 mice), 150 μg of SECs in phosphate buffered saline solution was infused. Mice in the NG-SEC conjugates group (n = 7 mice) received an infusion of 300 μΐ_^ of solution containing NG-SEC conjugates of the invention in an amount equivalent to 150 μg of DNA, infused at a rate of 600 μΐ_^ per minute. Thirteen days later, the mice were euthanized and transcardially perfused with only saline. The brain of the mouse was quickly removed and divided into the left and the right hemisphere, and the hippocampal region of each hemisphere was dissected away from the remaining brain tissue and frozen on dry ice. The relative amount of shRNA transcripts in the tissue samples was determined using the RT-PCR assay as previously described in Example 8, with the exception that the average ratio of shRNA transcripts to SNO RNA in the SEC group of mice was arbitrarily set to 1.0 in this experiment, because there is no saline only group in this experiment, unlike Example 8. Figure 13 shows that the brain samples of the left hemispheres of the mice treated with NG-SEC conjugates contained 2.35 times more shRNA transcripts relative to SNO RNA than mice receiving SECs alone. This observed difference is statistically significant at a level approaching traditional significance values (p < 0.0618). These results indicate that the NG-SEC conjugates of the invention are capable of delivering functional DNA comprising a Silencer Expression Cassette to the brain of a mammal upon administration via the carotid artery of the mammal. Example 15

Histone-DNA complexes help to mediate the transfection of DNA into Neuro2A cells [00196] In the present example, there are five histones that complex together to form nucleosomes. Four histones H2A, H2B, H3, and H4 form the "core" of the nucleosome, which is an octamer consisting of 3 of each of the core histones. HI, a linker histone, helps to keep the DNA wrapped around the core and stabilizes the chromatin structure. Each nucleosome condenses 146 base pairs of DNA, leaving some DNA as "linker DNA." This process is depicted in Figure 15. The 530 bp SEC1749 DNA would need three nucleosomes to condense completely. Figure 16 depicts multiple nucleosome core particles.

[00197] Neuro2A ("N2A") cells were plated in 6-well plates and treated with

SEC 1749 plus the commercial transfection reagent Transit-Neuro (Minis, Inc.), the G-N- SEC conjugate plus Transit-Neuro, the SEC alone, the G-N-SEC conjugate alone, the SEC mixed with histones, and the G-N-SEC conjugate mixed with histones. All of the treatments were administered with phosphate buffered saline (PBS) having pH 7.4. For the DNA and histone combinations, the DNA and histones were mixed to create either a charged neutral complex or a slightly positively charged complex. The actual histones used for this example were Histone Type II-A (unfractioned whole histones) (Sigma- Aldrich). Equal volumes of DNA and histone solution were mixed, and the histone solution concentration was varied to create different molar ratios of histones to DNA. The final volume of each treatment was kept constant at 250 μΐ_^ by adding an appropriate amount of PBS.

[00198] The cells were lysed after 48 hours, and RNA was isolated. PCR analysis was performed using cDNA made from the isolated RNA. The following calculations were made based on the concentration of the G-N-SEC conjugate. Because the concentration of the SEC is different than the G-N-SEC concentration, the histone to DNA molar ratios are not the same. Table 4 shows the G-N-SEC concentration data. TABLE 4

G-N-SEC

G-N-SEC concentration = 96.78 ng/μΕ. Table 5 shows the SEC concentration data.

TABLE 5

SEC

SEC concentration

[00199] The molar ratio for the histones to SEC 1749 was calculated using the charge ratio of histones to DNA. The charge ratio is as described by Balicki and Beutler (Histone H2A significantly enhances in vitro DNA transfection, MOLECULAR MEDICINE, 1997; 3(l l):782-787). The charge ratio of histones to DNA in this example was calculated to be 5.2(+): l(-). Using this charge ratio and the molecular weight of the nucleosomes, the molar ratio for the histones to SEC 1749 DNA was calculated to be 31: 1. This histone concentration needed for G-N-SEC was 1.136 μg/μL, and using this histone concentration, the final ratio of histones to SEC was 5.4:1.

[00200] The addition of histones to the SEC and to the G-N-SEC conjugate condenses and protects the DNA to improve its transfection and expression. Figure 17 shows the relative expression of SEC1749 in N2A cells treated with SEC1749 or G-N-SEC1749 with and without histones. As shown in the graph, the addition of histones to the SEC alone and to the G-N-SEC conjugate caused an increase in SEC 1749 expression in N2A cells. In cells treated with the 31: 1 molar ratio of histones to G-N-SEC conjugate, a dose response was observed, with a significant increase in transfection of the DNA. This molar ratio indicates about ten times the number of histones required to completely condense the DNA. There was also an increase in expression in N2A cells treated with a ratio of about two times the required number of histones to G-N-SEC DNA (5.8: 1). The expression for the 31: 1 ratio at the 5 μg and 10 μg doses were comparable or higher than the expression observed for the transfected positive controls at the 2 μg dose.

[00201] Treatment with the G-N-SEC conjugate alone did not show higher expression than treatment with SEC 1749 alone in the N2A cells. However, when histones were added, there was a significant increase in SEC 1749 expression in the N2A cells treated with the conjugate as compared to treatment with histones and SEC. The increase was best seen at the 31: 1 histones to G-N-SEC conjugate ratio. Condensing the DNA on histones thus increased the effect of the conjugated G-N peptide. In the negative control (i.e., cells treated with histones only), no SEC 1749 was measured above the background level of the assay.

[00202] This data is broken down into more detail in Figures 18-20. Figure 18 illustrates the effect of histones on the relative expression of SEC1749 in N2A cells treated with unmodified SEC1749. The N2A cells treated with a 5.4: 1 histones to SEC ratio show an increase in SEC1749 expression compared to N2A cells treated with the unmodified SEC alone. The data does not show a dose-dependent curve.

[00203] Figure 19 illustrates the effect of histones on the relative expression of

SEC1749 in N2A cells treated with the G-N-SEC1749 conjugate. The N2A cells treated with the combination of histones and G-N-SEC 1749 conjugate showed an increase in SEC expression compared to N2A cells treated with the conjugate alone. The expression of SEC1749 was the highest when histones were combined with a 10 μg dose of DNA in a 31: 1 molar ratio.

[00204] Figure 20 illustrates the effect of conjugating the G-N peptide to SEC1749, as demonstrated by the relative expression of SEC 1749 in treated N2A cells. The treatment using G-N-SEC combined with histones resulted in higher expression than the unmodified SEC mixed with histones. As shown in the graph, the conjugated peptide-polynucleotide increased expression 6-fold for the 10 μg treatment.

[00205] In this experiment, the cell lysate products were also assayed by RT-PCR to determine the effects of various treatments on the level of BACE1 expression in the N2A cells resulting from the transfection by SEC1749, which targets BACE1 mRNA. Figure 21 shows BACE1 suppression in N2A cells as determined by a measurement of BACE1 expression relative to controls. As shown in the graph, the best suppression of BACE1 was seen at the molar ratio of 15: 1 histones to G-N-SEC.

[00206] This experiment showed that histones aid in the transfection of DNA into N2A cells. In addition, the G-N peptide conjugation increases expression of the SEC/histones complex. Finally, the most efficacious treatment was the 31: 1 ratio for histone to G-N- SEC 1749 conjugate. This treatment resulted in the most significant increase in SEC1749 expression compared to the conjugate and SEC alone.

Example 16

Unfractioned Histone Experiment 2

[00207] Another experiment was conducted to see if the results of Example 15 could be replicated. Again, N2A cells were placed in 6-well plates and treated with SEC plus the commercial transfection reagent Transit- Neuro (Minis, Inc.), G-N-SEC 1749 plus the transfection reagent, unmodified SEC1749, G-N-SEC1749 alone, SEC1749 combined with histones, and G-N-SEC 1749 combined with histones. All treatments were administered in a

PBS buffer with pH 7.4.

[00208] This experiment supports the conclusions drawn in Example 15. The overall trend suggested a dose-dependent effect and an effect dependent upon the molar ratio of histones to DNA. Consistent with Example 15, a molar ratio of 30: 1 of histones to G-N- SEC 1749 DNA showed the highest SEC 1749 shRNA expression in N2A cells. The effect of histones on the expression of SEC 1749 in N2A cells is shown in Figure 22.

[00209] Figure 23 illustrates BACE1 suppression in N2A cells following treatment with SEC1749 and G-N-SEC1749, with or without histones, as determined by a measurement of BACE1 expression relative to controls. This BACE1 data suggests that the most effective treatment dose is either the 15: 1 molar ratio of histones to DNA with 5 μg G-N-SEC1749 conjugate or the 30: 1 molar ratio with 5 μg unmodified SEC1749.

[00210] To resolve this inconsistency, SEC1749 expression was then compared to mBACEl expression in N2A cells, as shown in Figure 24. Figure 25 further compares the expression of SEC1749 shRNA in N2A cells with the number of SEC1749 DNA copies. The graph in Figure 24 shows that the levels of SEC 1749 shRNA do not always correlate to mBACEl suppression. This may be partially explained by the large number of DNA copies in some of the samples, as shown in Figure 25.

[00211] The cells were further analyzed to examine SEC1749 shRNA expression as compared to BACE1 suppression for treatments using unmodified SEC 1749. The effect of histones on the expression of SEC1749 in N2A cells treated with unmodified SEC1749 and unmodified SEC 1749 combined with histones is shown in Figure 26. Figure 27 illustrates BACE1 suppression in N2A cells following treatment using unmodified SEC 1749 with histones. Figure 28 shows a comparison of SEC1749 shRNA and BACE1 expression in N2A cells treated with unmodified SEC 1749 with or without histones. Figure 29 further compares the expression of SEC1749 shRNA in N2A cells with the number of SEC1749 DNA copies. In the N2A cells treated with the unmodified SEC plus histones, the 30: 1 molar ratio of histones to DNA at a 10 μg dose of SEC1749 resulted in the highest shRNA expression. However, at this dose and molar ratio, the higher expression may have been due to the larger number of DNA copies found in that sample. In contrast, the mBACEl suppression data suggests that the best dose is 5 μg DNA at a 30: 1 molar ratio of histones to unmodified SEC 1749 DNA.

[00212] The same detailed analysis was performed for the treatments using the G-N-

SEC1749 conjugate. Figure 30 shows the effect of histones on the relative expression of SEC 1749 in N2A cells treated with the conjugate with or without histones. Figure 31 illustrates BACE1 suppression in N2A cells following treatment with G-N-SEC1749 with histones. The cells were again analyzed to examine SEC 1749 shRNA expression as compared to BACE1 suppression in N2A cells treated with the G-N-SEC1749 conjugate. Figure 32 shows a comparison of SEC1749 shRNA and BACE1 expression in N2A cells treated with G-N-SEC1749 with or without histones. Figure 33 further compares the expression of SEC1749 shRNA in N2A cells with the number of SEC1749 DNA copies. In N2A cells treated with the G-N-SEC1749 combined with histones, the 30: 1 molar ratio of histones to DNA at a 10 μg dose of SEC1749 resulted in the highest shRNA expression. However, at this dose and molar ratio, there was, again, a large number of DNA copies. Taking these copies into account, the 15: 1 molar ratio at 10 μg or the 5 μg dose at 30: 1 molar ratio may lead to higher expression of shRNA. The mBACEl suppression data suggests that the best doses are 5μg and 10μg at molar ratios of 6: 1 and 15: 1 histones to G-N-SEC1749 DNA.

[00213] Taken together, Examples 15 and 16 suggest that the best histone to DNA ratio for transfection is dependent on the DNA construct used. With unmodified SEC, the optimal histone to DNA ratio is 30: 1, and with the G-N-SEC conjugate, the optimal molar ratios having similar effects are 6: 1 and 15: 1. Due to likely DNA contamination from numerous DNA copies, it is more informative to screen for the mBACEl suppression first, rather than for shRNA transcript expression. DNA contamination in the samples leads to higher apparent expression of the SEC 1749 because they are detected by the shRNA assay. Therefore, to best understand the effect of the histones and modifying peptides, the desired outcome of BACE1 suppression is the most informative data.

[00214] It is to be understood that the present invention is not limited to the particular embodiments, materials, and examples described herein. In addition, the terminology used herein is used for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention.

Claims

WE CLAIM:

1. A peptide-polynucleotide composition having the formula

G-x-P-Y, or

P-x-G-Y

where

G is a rabies virus glycoprotein;

P is a domain having DNA condensing properties;

Y is a single or double stranded DNA, or single or double stranded RNA; and x is an optional linker comprising 0 to 100 glycines and/or polyethylene glycol (PEG).

2. The peptide-polynucleotide composition of claim 1, wherein at least one amino acid or nucleotide in the peptide-polynucleotide composition has azide or alkyne functionality.

3. The peptide-polynucleotide composition of claim 2, wherein the azide functionality comprises a polyethylene glycol (PEG) having a terminal azide group wherein the number of PEG repeat groups is from 1 to 150 and the alkyne functionality is a pho sphoramidite .

4. The peptide-polynucleotide composition of claim 1, wherein P is SEQ ID Nos.

2 or 31.

5. The peptide-polynucleotide composition of claim 1, wherein G is SEQ ID No. 1 or SEQ ID No. 81.

6. The peptide-polynucleotide composition of claim 1, wherein the peptide is any one of SEQ ID Nos. 3-4, 12 and 28-29.

7. The peptide-polynucleotide composition of claim 1, wherein PEG is from 2kDa to lOkDa.

8. The peptide-polynucleotide composition of claim 1, wherein Y is covalently linked to G-x, or Y is a plasmid that associates with the peptide.

9. The peptide-polynucleotide composition of claim 1, wherein Y is a Silencer Expression Cassette (SEC), comprising:

a promoter sequence wherein the promoter sequence is an RNA Polymerase II promoter selected from SEQ ID Nos. 34-36 or an RNA Polymerase III promoter selected from SEQ ID Nos. 32 and 33;

a sequence encoding shRNA selected from SEQ ID Nos. 66-80; and optionally,

a transcription termination sequence.

10. The peptide-polynucleotide composition of claim 9, wherein the SEC is SEQ ID Nos. 11, 30, or 38.

11. The peptide-polynucleotide composition of claim 1, wherein the DNA of the peptide-polynucleotide composition is condensed with histones in a molar ratio from n: 1 of histones to DNA, for each n={x£R | l<x<31 }.

12. A peptide-polynucleotide conjugate having the formula

G-x-N-Y, or

N-x-G-Y

where

G is a rabies virus glycoprotein;

N is a nuclear localization signal (NLS) amino acid sequence;

Y is a single or double stranded DNA, or single or double stranded RNA; and x is an optional linker comprising 0 to 100 glycines and/or polyethylene glycol (PEG).

13. The peptide-polynucleotide conjugate of claim 12, wherein at least one amino acid or nucleotide in the peptide-polynucleotide conjugate has azide or alkyne functionality.

14. The peptide-polynucleotide conjugate of claim 13, wherein the azide functionality comprises a polyethylene glycol (PEG) having a terminal azide group wherein the number of PEG repeat groups is from 1 to 150 and the alkyne functionality is a pho sphoramidite .

15. The peptide-polynucleotide conjugate of claim 12, wherein N is selected from any one of SEQ ID Nos. 5 and 64-65.

16. The peptide-polynucleotide conjugate of claim 12, wherein G is SEQ ID No. 1 or SEQ ID No. 81.

17. The peptide-polynucleotide conjugate of claim 12, wherein the peptide is any one of SEQ ID Nos. 9-10 and 15-18.

18. The peptide-polynucleotide conjugate of claim 12, wherein Y is a plasmid that associates with the peptide.

19. The peptide-polynucleotide conjugate of claim 12, wherein the PEG is from 2kDa to lOkDa.

20. The peptide-polynucleotide conjugate of claim 12, wherein Y is a Silencer Expression Cassette (SEC), comprising:

a promoter sequence wherein the promoter sequence is an RNA Polymerase II promoter selected from SEQ ID Nos. 34-36 or an RNA Polymerase III promoter selected from SEQ ID Nos. 32 and 33;

a sequence encoding shRNA selected from SEQ ID Nos. 66-80; and optionally,

a transcription termination sequence.

21. The peptide-polynucleotide conjugate of claim 20, wherein the SEC is SEQ ID Nos. 11, 30, or 38.

22. The peptide-polynucleotide conjugate of claim 12, wherein the single or double stranded DNA, or single or double stranded RNA of the peptide-polynucleotide conjugate is condensed with histones in a molar ratio from n: 1 of histones to DNA, for each n={ x£R I l<x<31 } .

23. A peptide-polynucleotide composition selected from any one of the formulas

where

G is a rabies virus glycoprotein;

P is a domain having DNA condensing properties;

N is a nuclear localization signal (NLS) amino acid sequence;

Y is a single or double stranded DNA, or single or double stranded RNA;

xi is an optional first linker comprising 0 to 100 glycines and/or polyethylene glycol (PEG); and

x₂ is an optional second linker comprising 0 to 100 glycines and/or polyethylene glycol (PEG).

24. The peptide-polynucleotide composition of claim 23, wherein at least one amino acid or nucleotide in the peptide-polynucleotide composition has azide or alkyne functionality.

25. The peptide-polynucleotide composition of claim 24, wherein the azide functionality comprises a polyethylene glycol (PEG) having a terminal azide group wherein the number of PEG repeat groups is from 1 to 150 and the alkyne functionality is a pho sphoramidite .

26. The peptide-polynucleotide composition of claim 23, wherein G is SEQ ID No. 1 or SEQ ID No. 81.

27. The peptide-polynucleotide composition of claim 23, wherein N is selected from SEQ ID Nos. 5 and 64-65.

28. The peptide-polynucleotide composition of claim 23, wherein P is SEQ ID Nos. 2 or 31.

29. The peptide-polynucleotide composition of claim 23, wherein the peptide is selected from any one of SEQ ID Nos. 6-8, 14, 20-27 and 39-63.

30. The peptide-polynucleotide composition of claim 23, wherein Y is covalently linked to G-x^N, or N-x^G, or Y is a plasmid that associates with the peptide.

31. The peptide-polynucleotide composition of claim 23, wherein Y is a Silencer Expression Cassette (SEC), comprising:

a promoter sequence wherein the promoter sequence is an RNA Polymerase II promoter selected from SEQ ID Nos. 34-36 or an RNA Polymerase III promoter selected from SEQ ID Nos. 32 and 33;

a sequence encoding shRNA selected from SEQ ID Nos. 66-80; and optionally,

a transcription termination sequence.

32. The peptide-polynucleotide composition of claim 31, wherein the SEC is SEQ ID Nos. 11, 30, or 38.

33. The peptide-polynucleotide composition of claim 23, wherein the single or double stranded DNA, or single or double stranded RNA of the peptide-polynucleotide conjugate is condensed with histones in a molar ratio from n: 1 of histones to DNA, for each n={x£R I l<x<31 }.

34. A method for transfecting a cell with DNA, comprising the steps of:

administering a peptide-polynucleotide composition in any one of claims 1-10 and 23-

32, or administering a peptide-polynucleotide conjugate in any one of claims 12-21 to a mammal, and

expressing the DNA in a target cell.

35. The method of claim 34, wherein the peptide-polynucleotide composition or conjugate is administered by injection to the carotid or femoral artery or intracranially or intraparenchymally.

36. The method of claim 35, where the method of intracranial or intraparenchymal administration is by injection or by a catheter in communication with a drug delivery device.

37. The method of claim 34, wherein the peptide-polynucleotide composition or conjugate is combined with a pharmaceutically acceptable buffer.

38. The method of claim 37, wherein the pharmaceutically acceptable buffer is an injectable saline solution or a phosphate buffered saline solution.

39. The method of claim 34, wherein another G-x or x-G peptide is conjugated to a 5 prime end of a complementary strand of Y.

40. The method of claim 34, wherein another G-x-N peptide is conjugated to a 5 prime end of a complementary strand of Y.

41. The method of claim 34, wherein the DNA of the peptide-polynucleotide composition is condensed with histones in a molar ratio from n: 1 of histones to DNA, for each n={x£R | l<x<31 }.

42. The method of claim 34, wherein another N-xrG peptide is conjugated to a 5 prime end of a complementary strand of Y.

43. A method of treating neurodegenerative disease, comprising the step of:

intracranially or intraparenchymally administering the peptide-polynucleotide composition in any of claims 1-10 and 23-32 or peptide-polynucleotide conjugate in any of claims 12-21 to a mammal in need thereof.

44. The method of claim 43, wherein the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, Huntington's disease, Lewy body dementia, a lysosomal storage disease, or Tay Sachs disease.

45. The method of claim 43, wherein the composition or conjugate is administered arterially or intravenously.

46. The method of claim 43, wherein the method of administration is by injection or by a catheter in communication with a drug delivery pump.

47. The method of claim 43, wherein Y codes for shRNA.

48. The method of claim 47, wherein the shRNA targets any of the messenger RNA molecules encoding for proteins selected from beta- amyloid cleaving enzyme type 1, gamma-secretase, amyloid precursor protein, a-synuclein, Huntingtin Protein, ataxin-1, ataxin-2, ataxin-3, atrophin-1, caspase-3, and caspase-9.

49. The method of claim 47, wherein the shRNA targets any of the RNA molecules that are transcription products of the genes BACEl, PSENl, APP, SNCA, HD, SCA1, SCA2, SCA3, DRPLA, CASP3, and CASP9.

50. A method of suppressing gene expression in target cells of a mammal, comprising the steps of:

administering a peptide-polynucleotide composition in any of claims 1-10 and 23-32, or the peptide-polynucleotide conjugate in any of claims 12-21 to the mammal.

51. The method of claim 50, wherein the composition or conjugate is administered intracranially, intraparenchymally, or intravenously, or by injection to the carotid or femoral artery.

52. The method of claim 51, wherein the method of intracranial, intraparenchymal or intravenous administration is by injection or by a catheter in communication with a drug delivery device.

53. A method of treating and/or delaying onset or worsening of a symptom in a mammal with or at risk of neurodegenerative disease, comprising the steps of:

intracranially or intraparenchymally administering naked siRNA, the peptide- polynucleotide composition in any of claims 1-10 and 23-32, or the peptide-polynucleotide conjugate in any of claims 12-21, to the mammal.

54. The method of claim 53, wherein the method of administration is by injection or by a catheter in communication with a drug delivery pump.

55. The method of claim 53, wherein the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, Huntington's disease, Lewy body dementia, a lysosomal storage disease, or Tay Sachs disease.

56. The method of claim 53, wherein Y codes for shRNA.

57. The method of claim 56, wherein the shRNA targets any of the messenger RNA molecules encoding for proteins selected from beta- amyloid cleaving enzyme type 1, gamma-secretase, amyloid precursor protein, a-synuclein, Huntingtin Protein, ataxin-1, ataxin-2, ataxin-3, atrophin-1, caspase-3, and caspase-9.

58. The method of claim 56, wherein the shRNA targets any of the RNA molecules that are transcription products of the genes BACEl, PSENl, APP, SNCA, HD, SCA1, SCA2, SCA3, DRPLA, CASP3, and CASP9.

59. A method of making a peptide-polynucleotide conjugate, comprising the steps of:

modifying an amino acid of the peptide to have azide functionality,

modifying a nucleotide of the polynucleotide to have alkyne functionality, and reacting the azide and alkyne functionality to form a 5-membered heterocycle (triazole ring) using a copper catalyst to covalently bond the peptide to the polynucleotide.

60. The method of claim 59, further comprising the step of stabilizing the copper catalyst using tris-(benzyltriazolylmethyl)amine (TBTA).

61. The method of claim 60, wherein TBTA is used in a molar excess of 2 to 1 compared to the copper catalyst.

62. The method of claim 59, wherein the amino acid is modified with NHS- (PEO)₄-azide.

63. The method of claim 59, wherein the nucleotide is modified using an alkyne functional phosphoramidite.

64. The method of claim 63, wherein the alkyne functional phosphoramidite is 5'- hexynyl phosphoramidite.

65. The method of claim 59, wherein the azide functionality is selected from the group consisting of azidohomoalanine, asidoalanine, 2-amino-5-hexanoic acid,

azidophenylalanine, and alkynyl tyrosine.

66. A method of increasing shRNA expression in target cells of a mammal, comprising the steps of:

administering the peptide-polynucleotide composition in any of claims 1-10 and 23- 32, or the peptide-polynucleotide conjugate in any of claims 12-21.

67. The method of claim 66, wherein the composition or conjugate is administered intracranially, intraparenchymally, intravenously or to the carotid or femoral artery.

68. The method of claim 67, where the method of administration is by injection or by a catheter in communication with a drug delivery pump.

69. The method of claim 66, further comprising any one of SEQ ID Nos. 11, 30, or 38.

70. The method of claim 66, wherein the shRNA targets any of the RNA molecules that are transcription products of the genes BACEl, PSENl, APP, SNCA, HD, SCA1, SCA2, SCA3, DRPLA, CASP3, and CASP9.

71. The method of claim 69, wherein the DNA in the composition or conjugate is mixed with histones in a molar ratio from n: 1 of histones to DNA, for each

n={x£R I l<x<31 }.

72. The method of claim 66, wherein the conjugate is one in any one of claims 20- 21 and is mixed with histones in a ratio of 15: 1 or 6: 1.

73. A method of enhancing siRNA uptake by target cells of a mammal comprising administering via the carotid artery of a mammal a peptide-polynucleotide conjugate having the formula

G-x -Y,

where

G is a rabies virus glycoprotein;

Y is a single or double stranded DNA, or single or double stranded RNA; and x is an optional linker comprising 0 to 100 glycines and/or polyethylene glycol (PEG).

74. The method of claim 73, wherein Y is siRNA.

75. The method of claim 73, wherein Y is a Silencer Expression Cassette (SEC).

76. The method of claim 73, wherein the SEC is SEQ ID Nos. 11, 30, or 38.

77. The method of claim 73, wherein the DNA is mixed with histones in a molar ratio from n: l of histones to DNA, for each n={x£R | l<x<31 }.

78. The method of claim 73, wherein the composition or conjugate is administered intracranially, intraparenchymally, arterially to the carotid or femoral artery or intravenously.

79. The method of claim 78 wherein the method of administration is by injection or by a catheter in communication with a drug delivery pump.