Regulatory polynucleotides

Abstract

The present invention relates to regulatory polynucleotides, in particular, the present invention relates to compositions and methods of making, producing and therapeutic uses of regulatory polynucleotides.

Description

[0001] This application is a divisional application of the Chinese patent application with application number 201580073443.6 (2020113129942), application date November 13, 2015, and invention name “Regulatory Polynucleotides”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 079,590, entitled Modulatory Polynucleotides, filed on November 14, 2014, U.S. Provisional Patent Application No. 62 / 212,004, entitled Modulatory Polynucleotides, filed on August 31, 2015, and U.S. Provisional Patent Application No. 62 / 234,477, entitled Modulatory Polynucleotides, filed on September 29, 2015; the contents of each of which are incorporated herein by reference in their entirety.

[0004] Reference to a sequence listing

[0005] This application is filed with a sequence listing in electronic form. The sequence listing is provided as a file titled 1014PCTSL.txt created on November 12, 2015, which is 235,330 bytes in size. The information in the electronic sequence listing is incorporated herein by reference in its entirety. Technical Field

[0006] The present invention relates to compositions, methods, processes, kits and devices for designing, preparing, producing and / or formulating regulatory polynucleotides. In certain embodiments, such regulatory polynucleotides may be encoded by or within a recombinant adeno-associated virus (AAV) and may comprise artificial microRNAs (microRNAs), artificial pre-microRNAs (pre-microRNAs) and / or artificial primary-microRNAs (pri-microRNAs). Background Art

[0007] MicroRNA (or miRNA or miR) is small, non-coding, single-stranded ribonucleic acid molecule (RNA), which is often 19-25 nucleotides in length. More than 1000 kinds of microRNAs have been identified in mammalian genomes. Mature microRNAs mainly bind to the 3' untranslated region (3'-UTR) of target messenger RNA (mRNA) by partially or completely pairing with the complementary sequence of target mRNA, thereby promoting the degradation of target mRNA at the post-transcriptional level, and in some cases, inhibiting the initiation of translation. MicroRNA plays a key role in many key biological processes (such as cell cycle and growth regulation, apoptosis, cell proliferation and tissue development).

[0008] miRNA genes are usually transcribed as long primary transcripts of miRNA (i.e., primary-miRNA (pri-miRNA)).Primary-miRNA is cleaved into miRNA precursors (i.e., pre-miRNA (pre-miRNA)), which are further processed to produce mature and functional miRNA.

[0009] Although many target expression strategies employ nucleic acid-based modalities, there remains a need for improved nucleic acid modalities with greater specificity and fewer off-target effects.

[0010] The present invention provides such improved modalities in the form of artificial primary-, pre- and mature microRNA constructs and their design methods. These novel constructs can be synthetic independent molecules, or encoded in plasmids or expression vectors for delivery to cells. Such vectors include but are not limited to adeno-associated virus vectors such as vector genomes of any AAV serotype, or other viral delivery vehicles such as slow viruses, etc. Summary of the invention

[0011] Described herein are compositions, methods, processes, kits and apparatuses for designing, preparing, producing and / or formulating regulatory polynucleotides.

[0012] In certain embodiments, such regulatory polynucleotides may be encoded by or contained within a plasmid or vector or a recombinant adeno-associated virus (AAV), and may comprise an artificial microRNA, an artificial pre-microRNA and / or an artificial primary-microRNA.

[0013] The details of various embodiments of the invention are set forth in the following description. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The foregoing and other objects, features and advantages will become apparent from the following description of specific embodiments of the invention as illustrated in the accompanying drawings, in which like reference numerals represent like parts in different views. The drawings are not necessarily to scale, emphasis instead being placed upon explaining the principles of the various embodiments of the invention.

[0015] Figure 1 is a schematic diagram of an artificial primary-microRNA encoded in an AAV vector according to the present invention.

[0016] Figure 2 The histogram shows the activity of primary-mRNA constructs encoded in AAV vectors.

[0017] Figure 3 The histogram shows the activity of the guide strand of the regulatory polynucleotide encoded in the AAV vector in HEK293T cells.

[0018] Figure 4 The histogram shows the activity of the passenger strand of the regulatory polynucleotide encoded in the AAV vector in HEK293T cells.

[0019] Figure 5 The histogram shows the activity of the guide strand of the regulatory polynucleotide encoded in the AAV vector in HeLa cells.

[0020] Figure 6 The histogram shows the activity of the passenger strand of the regulatory polynucleotide encoded in the AAV vector in HeLa cells.

[0021] Figure 7 is a histogram showing the quantification of intracellular AAV DNA expressed.

[0022] Figure 8 The histogram shows the activity of constructs encoded in AAV vectors in human motor neurons.

[0023] Fig. 9 The graph shows dose-dependent SOD1 silencing in U251MG cells.

[0024] Fig.10 The graph shows dose-dependent SOD1 silencing in human astrocytes.

[0025] Fig.11 The graph shows the time course of SOD1 silencing in U251MG cells.

[0026] Figure 12 contains Fig. 12A , Fig. 12B and Fig. 12C , which are graphs showing the dose-dependent effects of the constructs. Fig. 12ARelative SOD1 expression is shown. Fig. 12B The percentage of the guide chain is shown. Fig. 12C The percentage of passenger chains is shown.

[0027] Fig.13 A simplified diagram shows the position of the regulatory polynucleotide (MP) relative to the ITR, intron (I) and poly(A) (P). DETAILED DESCRIPTION

[0028] Compositions of the present invention

[0029] According to the present invention, there is provided a regulatory polynucleotide that works as an artificial microRNA. "Regulatory polynucleotide" used herein is any nucleic acid polymer that works to regulate the level or amount of (increase or decrease) target gene. Regulatory polynucleotides are included in precursor molecules processed in cells before regulation. Regulatory polynucleotides or their processed forms can be encoded in plasmids, vectors, genomes or other nucleic acid expression vectors for delivery to cells.

[0030] In certain embodiments, regulatory polynucleotides are designed as primary microRNAs (primary-miRs) or precursor microRNAs (pre-miRs), which are processed within the cell to produce highly specific artificial microRNAs.

[0031] Based on the sequence or structural support of classical (canonical) or known microRNA, elementary-microRNA or pre-microRNA, regulatory polynucleotides, particularly artificial microRNA of the present invention can be designed.Such a sequence can correspond to any known microRNA or its precursor, such as those taught in US2005 / 0261218 and US2005 / 0059005 (its content is incorporated herein by reference in its entirety) disclosed in the U.S.

[0032] MicroRNA (or miRNA or miR) is a non-coding RNA of 19-25 nucleotides in length that binds to the 3'UTR of a nucleic acid molecule and downregulates gene expression (either by reducing nucleic acid molecule stability or by inhibiting translation). The regulatory polynucleotides of the present invention may comprise one or more microRNA sequences, microRNA seeds or artificial microRNAs, e.g., sequences that function as microRNAs.

[0033] The microRNA sequence comprises a "seed" region, i.e., a sequence in the region of position 2-9 of a mature microRNA, which has perfect Watson-Crick complementarity with the miRNA target sequence. The microRNA seed may comprise position 2-8 or 2-7 or 2-9 of a mature microRNA. In certain embodiments, the microRNA seed may comprise 7 nucleotides (e.g., nucleotides 2-8 of a mature microRNA), wherein the seed complementary site in the corresponding miRNA target is flanked by an adenine (A) relative to microRNA position 1. In certain embodiments, the microRNA seed may comprise 6 nucleotides (e.g., nucleotides 2-7 of a mature microRNA), wherein the seed complementary site in the corresponding miRNA target is flanked by an adenine (A) relative to microRNA position 1. See, e.g., Grimson A, Farh KK, Johnston WK, Garrett-Engele P, Lim LP, Bartel DP; Mol Cell. July 6, 2007; 27 (1): 91-105; each of which is incorporated herein by reference in its entirety. In naturally occurring microRNAs, the bases of the microRNA seed have complete complementarity with the target sequence.

[0034] As taught herein, design parameters or rules have been identified and applied to design regulatory polynucleotides (eg, artificial microRNAs) with superior target gene regulatory properties and limited off-target effects.

[0035] In one embodiment, the molecular scaffolds of the regulatory polynucleotides described herein can be designed and optimized to create regulatory polynucleotides with desired target gene regulatory properties. As a non-limiting example, the regulatory polynucleotides can have excellent target gene regulatory properties and limited off-target effects.

[0036] In one embodiment, the regulatory polynucleotides of the present invention (such as artificial miR) comprise modular elements or sequence motifs assembled according to a set of rules, which result in highly specific target recognition and low guide / passenger ratios. Such modules or sequence motifs include, but are not limited to, double-stranded regions, flanking regions, loops, optimized loops, UGUG loops, GU domains, spacers (to control proximal and distal motifs or module spacing, or to introduce structural elements such as turns, loops or projections), CNNC motifs, and thermodynamically asymmetric regions, which may include loops, projections, mispairings, swings, and / or combinations thereof. Non-limiting examples of rules that can be applied alone or in combination when constructing artificial miRs include those taught in the following literature: Seitz et al. Silence 2011, 2:4; Gu, et al., Cell 151, 900-911, November 9, 2012; Schwartz, et al., Cell, Vol. 115, 199-208, October 17, 2003; Park, et al., Nature, Vol. 475, 101, July 14, 2011; Ketley et al., 2013, PLoS ONE 8(6); Liu, et al., Nucleic Acids Research, 2008, Vol. 36, No. 9 2811-2824; Dow, et al., 2013, Nat Protoc.; 7(2):374-393. doi:10.1038 / nprot.2011.446; Auyeung, et al., Cell 152, 844-858, February 14, 2013; Gu et al., Cell 2012 November 9, 151(4):900-11; Fellmann et al. Molecular Cell 41, 733-746, 2011; Han et al. Cell 125, 887-907, 2006; Betancur et al. Frontiers in Genetics, Vol. 3, Art. 127, July 1-6, 2012; Schwarz et al. Cell Vol. 115, 199-208, 2003; the contents of each of which are incorporated herein by reference in their entirety.

[0037] In addition to the module or sequence motif, the regulatory polynucleotide further comprises at least one or both of a passenger and a guide strand. The passenger and guide strand may be positioned or located on the 5' arm or the 3' arm of the stem-loop structure of the regulatory polynucleotide.

[0038] In one embodiment, the 3' stem arm of the regulatory polynucleotide may have 11 nucleotides downstream of the 3' end of the guide strand that are complementary to 11 of the 13 nucleotides upstream of the 5' end of the passenger strand in the 5' stem arm.

[0039] In one embodiment, the regulatory polynucleotide may have a cysteine ​​6 nucleotides downstream from the 3' end of the 3' stem arm of the regulatory polynucleotide.

[0040] In one embodiment, the regulatory polynucleotide comprises a miRNA seed match for the guide strand. In another embodiment, the regulatory polynucleotide comprises a miRNA seed match for the passenger strand. In another embodiment, the regulatory polynucleotide does not comprise a seed match for the guide strand or the passenger strand.

[0041] In one embodiment, the regulatory polynucleotide may have almost no significant full-length off-target of the guide strand. In another embodiment, the regulatory polynucleotide may have almost no significant full-length off-target of the passenger strand. In another embodiment, the regulatory polynucleotide may have almost no significant full-length off-target of the guide strand or the passenger strand.

[0042] In one embodiment, the regulatory polynucleotide may have high in vitro activity. In another embodiment, the regulatory polynucleotide may have low in vitro activity. In another embodiment, the regulatory polynucleotide may have high guide strand activity and low passenger strand activity in vitro.

[0043] In one embodiment, the regulatory polynucleotide has high guide strand activity and low passenger strand activity in vitro. The target knockdown (KD) achieved by the guide strand can be at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5% or 100%. The target knockdown achieved by the guide strand can be 60-65%, 60-70%, 60-75%, 60-80%, 60-85%, 60-90%, 60-95%, 60-99%, 60-99.5%, 60-100%, 65-70%, 65-75%, 65-80%, 65-85%, 65-90%, 65-95%, 65-99%, 65-99.5%, 65-100%, 70-75%, 70-80%, 70-85%, 70-90%, 70-95%, 70-99%, 70-99.5%, 70-100%, 75 In some embodiments, the target knockdown (KD) achieved by the guide strand is greater than 70%.

[0044] In one embodiment, the passenger strand is related to the IC of the most recent off-target 50 Greater than the IC of the guide strand on the target 50 As a non-limiting example, if the passenger strand is about the nearest off-target IC 50 Greater than the IC of the guide strand on the target 50 If the amount of the regulatory polynucleotide is 100 times that of the regulatory polynucleotide, then the regulatory polynucleotide is said to have high guide strand activity and low passenger strand activity in vitro.

[0045] In one embodiment, 5' processing of the guide strand has the following correct start (n) at the 5' end: at least 75%, 80%, 85%, 90%, 95%, 99% or 100% of the time in vitro or in vivo. As a non-limiting example, 5' processing of the guide strand is precise and has the following correct start (n) at the 5' end: at least 99% of the time in vitro. As a non-limiting example, 5' processing of the guide strand is precise and has the following correct start (n) at the 5' end: at least 99% of the time in vivo.

[0046] In one embodiment, the guide strand to passenger strand (G:P) ratio is 1:99, 5:95, 10:90, 15:85, 20:80, 25:75, 30:70, 35:65, 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 85:15, 90:10, 95:5, or 99: 1 in vitro or in vivo. As a non-limiting example, the guide strand to passenger strand ratio is 80:20 in vitro. As a non-limiting example, the guide strand to passenger strand ratio is 80:20 in vivo.

[0047] In one embodiment, the integrity of the vector genome is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more than 99% of the full length of the construct.

[0048] Regulatory polynucleotides

[0049] In one embodiment, any known RNAi construct or RNAi agent can serve as a starting construct for designing the passenger and / or guide strand of the regulatory polynucleotide or artificial microRNA of the present invention. These include classical siRNA, small interfering RNA (siRNA), double-stranded RNA (dsRNA), inverted repeats, short hairpin RNA (shRNA), small temporally regulatory RNA (stRNA), clustered inhibitory RNA (cRNA) (including radial clustered inhibitory RNA, asymmetric clustered inhibitory RNA, linear clustered inhibitory RNA and complex or composite clustered inhibitory RNA), dicer substrates, DNA-guided RNAi (ddRNAi), single-stranded RNAi (ssRNAi), microRNA (miRNA) antagonists, microRNA mimics, microRNA agonists, blockmirs (also known as Xmirs), microRNA mimetics, microRNA addbacks, supermiR, oligomeric constructs disclosed in PCT Publication WO / 2005 / 013901 (the contents of which are incorporated herein in their entirety), oligomeric constructs such as those disclosed in U.S. Publication No. 20090131 360 (the contents of which are incorporated herein in their entirety), the single-rxRNA constructs disclosed in PCT Publication WO / 2010 / 011346 (the contents of which are incorporated herein in their entirety); the sd-rxRNA constructs disclosed in PCT Publication WO / 2010 / 033247 (the contents of which are incorporated herein in their entirety), the dual-RNAi constructs that reduce RNA levels and also modulate immune responses (disclosed in PCT Publication WO / 2010 / 00 2851 and WO / 2009 / 141146, the contents of which are incorporated herein by reference in their entireties), and antigenic RNA (agRNA) or small activating RNA (saRNA) that increases the expression of the target for which they are designed (disclosed in PCT publications WO / 2006 / 130201, WO / 2007 / 086990, WO / 2009 / 046397, WO / 2009 / 149182, WO / 2009 / 086428, the contents of which are incorporated herein by reference in their entireties).

[0050] Likewise, any of the pre- or pre-microRNA precursors of the microRNAs listed above may also serve as molecular scaffolds for the regulatory polynucleotides of the present invention.

[0051] In one embodiment, the starting construct can be derived from any relevant species, such as, but not limited to, mouse, rat, dog, monkey, or human.

[0052] In one embodiment, the regulatory polynucleotide can be located in an expression vector downstream of a promoter (for example, but not limited to, CMV, U6, CBA or a CBA promoter with an SV40 intron). In addition, the regulatory polynucleotide can also be located upstream of a polyadenylation sequence in an expression vector. As a non-limiting example, the regulatory polynucleotide can be located in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more than 30 nucleotides downstream of a promoter and / or upstream of a polyadenylation sequence in an expression vector. As another non-limiting example, the regulatory polynucleotide can be located within 1-5, 1-10, 1-15, 1-20, 1-25, 1-30, 5-10, 5-15, 5-20, 5-25, 5-30, 10-15, 10-20, 10-25, 10-30, 15-20, 15-25, 15-30, 20-25, 20-30 or 25-30 nucleotides downstream of the promoter and / or upstream of the polyadenylation sequence in the expression vector. As a non-limiting example, the regulatory polynucleotide can be located within the first 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25% or more than 25% of the nucleotides downstream of the promoter and / or upstream of the polyadenylation sequence in the expression vector. As another non-limiting example, the regulatory polynucleotide may be located within the first 1-5%, 1-10%, 1-15%, 1-20%, 1-25%, 5-10%, 5-15%, 5-20%, 5-25%, 10-15%, 10-20%, 10-25%, 15-20%, 15-25% or 20-25% of nucleotides downstream of the promoter and / or upstream of the polyadenylation sequence in the expression vector.

[0053] In one embodiment, the regulatory polynucleotide can be located in the upstream of the polyadenylation sequence in the expression vector. In addition, the regulatory polynucleotide can be located in the downstream of a promoter (for example, but not limited to, CMV, U6, CBA or CBA promoter with SV40 intron) in the expression vector. As a non-limiting example, the regulatory polynucleotide can be located in the expression vector 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30 or more than 30 nucleotides downstream of the promoter and / or upstream of the polyadenylation sequence. As another non-limiting example, the regulatory polynucleotide can be located within 1-5, 1-10, 1-15, 1-20, 1-25, 1-30, 5-10, 5-15, 5-20, 5-25, 5-30, 10-15, 10-20, 10-25, 10-30, 15-20, 15-25, 15-30, 20-25, 20-30 or 25-30 nucleotides downstream of the promoter and / or upstream of the polyadenylation sequence in the expression vector. As a non-limiting example, the regulatory polynucleotide can be located within the first 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25% or more than 25% of the nucleotides downstream of the promoter and / or upstream of the polyadenylation sequence in the expression vector. As another non-limiting example, the regulatory polynucleotide may be located within the first 1-5%, 1-10%, 1-15%, 1-20%, 1-25%, 5-10%, 5-15%, 5-20%, 5-25%, 10-15%, 10-20%, 10-25%, 15-20%, 15-25% or 20-25% of nucleotides downstream of the promoter and / or upstream of the polyadenylation sequence in the expression vector.

[0054] In one embodiment, the regulatory polynucleotide may be located in a scAAV.

[0055] In one embodiment, the regulatory polynucleotide may be located in a ssAAV.

[0056] In one embodiment, the regulatory polynucleotide may be located near the 5' end of the flip ITR in the expression vector. In another embodiment, the regulatory polynucleotide may be located near the 3' end of the flip ITR in the expression vector. In another embodiment, the regulatory polynucleotide may be located near the 5' end of the flop ITR in the expression vector. In another embodiment, the regulatory polynucleotide may be located near the 3' end of the flop ITR in the expression vector. In one embodiment, the regulatory polynucleotide may be located between the 5' end of the flip ITR and the 3' end of the flop ITR in the expression vector. In one embodiment, the regulatory polynucleotide may be located between the 3' end of the flip ITR and the 5' end of the flip ITR in the expression vector (e.g., between the 5' end of the flip ITR and the 3' end of the flop ITR or midway between the 3' end of the flop ITR and the 5' end of the flip ITR). As a non-limiting example, the regulatory polynucleotide can be located within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more 30 nucleotides downstream of the 5' or 3' end of the ITR (e.g., Flip or Flop ITR) in the expression vector. As a non-limiting example, the regulatory polynucleotide can be located within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more 30 nucleotides upstream of the 5' or 3' end of the ITR (e.g., Flip or Flop ITR) in the expression vector. As further non-limiting examples, the regulatory polynucleotide may be located within 1-5, 1-10, 1-15, 1-20, 1-25, 1-30, 5-10, 5-15, 5-20, 5-25, 5-30, 10-15, 10-20, 10-25, 10-30, 15-20, 15-25, 15-30, 20-25, 20-30, or 25-30 nucleotides downstream of the 5' or 3' end of the ITR (e.g., Flip or Flop ITR) in the expression vector.As another non-limiting example, the regulatory polynucleotide can be located in the expression vector within 1-5, 1-10, 1-15, 1-20, 1-25, 1-30, 5-10, 5-15, 5-20, 5-25, 5-30, 10-15, 10-20, 10-25, 10-30, 15-20, 15-25, 15-30, 20-25, 20-30 or 25-30 nucleotides of the 5' or 3' end upstream of the ITR (e.g., Flip or Flop ITR). As a non-limiting example, the regulatory polynucleotide can be located in the expression vector within the first 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25% or more than 25% of the nucleotides of the 5' or 3' end upstream of the ITR (e.g., Flip or Flop ITR). As a further non-limiting example, the regulatory polynucleotide may be located within the first 1-5%, 1-10%, 1-15%, 1-20%, 1-25%, 5-10%, 5-15%, 5-20%, 5-25%, 10-15%, 10-20%, 10-25%, 15-20%, 15-25% or 20-25% of nucleotides downstream of the 5' or 3' end of the ITR (e.g., Flip or Flop ITR) in the expression vector.

[0057] Molecular scaffold

[0058] In certain embodiments, the initial molecular scaffold of the regulatory polynucleotide is known or wild-type primary-or pre-microRNA. In other embodiments, the molecular scaffold of the regulatory polynucleotide is designed from scratch (see Cullen, Gene Therapy (2006) 13,503-508, about miR30; Chung, et al., Nucleic Acids Research, 2006, Vol. 34, No. 7, about miR-155; its contents are incorporated herein by reference as a whole).

[0059] As used herein, a "molecular scaffold" is a framework or starting molecule that forms the structural basis for the sequence or antibody of a subsequent molecule to be designed or prepared.

[0060] Steering Figure 1 The regulatory polynucleotides of the present invention can be designed as primary-miRs as shown. In the figure, a primary-miR molecular scaffold is shown. The regulatory polynucleotide containing a payload (e.g., siRNA, miRNA or other RNAi agent described herein) contains a leading 5' flanking sequence, which can be of any length and can be derived entirely or partially from a wild-type microRNA sequence or be completely artificial.

[0061] Likewise, the 3' flanking sequence shown in the figure may map the 5' flanking sequence in size and origin. Either flanking sequence may be absent. The 3' flanking sequence may optionally contain one or more CNNC motifs, where "N" represents any nucleotide.

[0062] At least one payload sequence forms the stem of the stem-loop structure shown. In certain embodiments, the payload sequence comprises at least one nucleic acid sequence that is partially complementary to the target sequence and will hybridize. In certain embodiments, the payload is a wild-type microRNA. In certain embodiments, the payload is a siRNA molecule or a fragment of a siRNA molecule. In certain embodiments, the payload is a substantially double-stranded construct that may comprise one or more microRNAs, artificial microRNAs, or siRNAs.

[0063] In certain embodiments, the 5' arm of the stem loop comprises a passenger strand. This strand is also referred to as the sense strand because it reflects identity with the target. The passenger strand can be 15-30 nucleotides in length. It can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0064] In certain embodiments, the 3' arm of the stem loop comprises a guide strand. This strand is also referred to as an antisense strand because it reflects homology to the target. The guide strand can be a length of 15-30 nucleotides, a length of 21-25 nucleotides, or a length of 22 nucleotides. It can be a length of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some cases, the guide strand comprises a "G" nucleotide at the 5' end.

[0065] In certain embodiments, wherein the guide strand comprises microRNA or artificial microRNA, the guide strand may comprise one or more microRNA seed sequences. Relative to the first 5' nucleotide of the guide strand or relative to the dicer cleavage site, the seed sequence may be located at position 2-7, 2-8 or 2-9 of the guide strand.

[0066] In other embodiments, the passenger strand may be present on the 3' arm, while the guide strand is present on the 5' arm of the stem of the stem-loop structure.

[0067] The passenger and guide strands may be fully complementary over a majority of their lengths. In other embodiments, the passenger and guide strands may independently be at least 70%, 80%, 90%, 95%, or 99% complementary over at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% of the lengths of the strands.

[0068] Neither the identity of the passenger strand nor the homology of the guide strand need to be 100% complementary to the target.

[0069] The loop (also referred to as the loop motif) separates the passenger and guide strands of the stem-loop structure. The loop can be any length of 4-30 nucleotides, 4-20 nucleotides, 4-15 nucleotides, 5-15 nucleotides, 6-12 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides and / or 12 nucleotides.

[0070] In certain embodiments, the loop comprises at least one UGUG motif. In certain embodiments, the UGUG motif is located at the 5' end of the loop.

[0071] A spacer region may be present in a regulatory polynucleotide to separate one or more modules from each other. There may be one or more such spacer regions.

[0072] In one embodiment, a spacer region of 8-20 (i.e., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) nucleotides may be present between the passenger strand and the flanking sequence.

[0073] In one embodiment, the spacer is 13 nucleotides and is located between the 5' end of the passenger strand and the flanking sequence. In one embodiment, the spacer is of sufficient length to form approximately one helical turn of the sequence.

[0074] In one embodiment, a spacing region of 8-20 (i.e., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) nucleotides may be present between the guide strand and the flanking sequence.

[0075] In one embodiment, the spacer sequence is 10-13 (ie, 10, 11, 12 or 13) nucleotides and is located between the 3' end of the guide strand and the flanking sequence. In one embodiment, the spacer has sufficient length to form approximately one helical turn of the sequence.

[0076] In one embodiment, the regulatory polynucleotide comprises at least one UG motif at the base of the stem, wherein the G nucleotide is paired and the U nucleotide is unpaired. In certain embodiments, the unpaired U nucleotide is located in the flanking sequence.

[0077] In one embodiment, the regulatory polynucleotide comprises a 5' flanking sequence, a 5' arm, a loop motif, a 3' arm, and a 3' flanking sequence in the 5' to 3' direction. As a non-limiting example, the 5' arm may comprise a passenger strand, and the 3' arm comprises a guide strand. In another non-limiting example, the 5' arm comprises a guide strand, and the 3' arm comprises a passenger strand.

[0078] In one embodiment, the 5' arm, payload (e.g., passenger and / or guide strand), loop motif, and / or 3' arm sequence can be altered (e.g., replacing 1 or more nucleotides, adding nucleotides, and / or deleting nucleotides). Such alterations can result in beneficial changes in construct function (e.g., increasing knockdown of the target sequence, reducing degradation of the construct, reducing off-target effects, increasing the efficiency of the payload, and reducing degradation of the payload).

[0079] In one embodiment, the passenger strand sequence can be altered (e.g., 1 or more nucleotides are substituted, nucleotides are added, and / or nucleotides are deleted). As a non-limiting example, the passenger strand sequence can include 1 or 2 substitutions within the last 4 nucleotides of the sequence (e.g., C for G). As another non-limiting example, the passenger strand sequence can include 1 or 2 substitutions within the 7-15 nucleotides at the 5' end of the sequence (e.g., U for A, or C for G).

[0080] In one embodiment, the 3' arm sequence can be altered (e.g., 1 or more nucleotides are substituted, nucleotides are added, and / or nucleotides are deleted). As a non-limiting example, the sequence of the 3' arm can include 1 or 2 substitutions within the first 4 nucleotides of the sequence (e.g., A for U).

[0081] In one embodiment, the molecular scaffold of the payload construct may include a 5' flanking region, a loop motif, and a 3' flanking region. Between the 5' flanking region and the loop motif may be a first payload region, and between the loop motif and the 3' flanking region may be a second payload region. The first and second payload regions may include siRNA, miRNA, or other RNAi agents, fragments, or variants described herein. The first and second payload regions may also include sequences that are identical, different, or complementary to each other. As a non-limiting example, the first payload region sequence may be the passenger strand of the siRNA construct, and the second payload region sequence may be the guide strand of the siRNA construct. The passenger and guide sequences may be substantially complementary to each other. As another non-limiting example, the first payload region sequence may be the guide strand of the siRNA construct, and the second payload region sequence may be the passenger strand of the siRNA construct. The passenger and guide sequences may be substantially complementary to each other.

[0082] In one embodiment, the molecular scaffolds of the regulatory polynucleotides described herein comprise a 5' flanking region, a loop region, and a 3' flanking region. Non-limiting examples of sequences of the 5' flanking region, the loop region, and the 3' flanking region that can be used in the molecular scaffolds described herein are shown in Tables 1-3.

[0083] Table 1. 5' flanking regions of molecular scaffolds

[0084]

[0085]

[0086] Table 2. Loop motif regions of molecular scaffolds

[0087]

[0088] Table 3. 3' flanking regions of molecular scaffolds

[0089]

[0090]

[0091] Any of the regions described in Tables 1-3 may be used in the molecular scaffolds described herein.

[0092] In one embodiment, the molecular scaffold may comprise a 5' flanking region listed in Table 1. As a non-limiting example, the molecular scaffold may comprise a 5' flanking region 5F1, 5F2, 5F3 or 5F4.

[0093] In one embodiment, the molecular scaffold may comprise a loop motif region listed in Table 2. As a non-limiting example, the molecular scaffold may comprise a loop motif region L1, L2, L3, L4 or L5.

[0094] In one embodiment, the molecular scaffold may comprise a 3' flanking region listed in Table 3. As a non-limiting example, the molecular scaffold may comprise a 3' flanking region 3F1, 3F2, 3F3, 3F4, 3F5 or 3F6.

[0095] In one embodiment, the molecular scaffold may comprise at least one 5' flanking region and at least one loop motif region described in Tables 1 and 2. As a non-limiting example, the molecular scaffold may comprise 5F1 and L1, 5F1 and L2, 5F1 and L3, 5F1 and L4, 5F1 and L5, 5F2 and L1, 5F2 and L2, 5F2 and L3, 5F2 and L4, 5F2 and L5, 5F3 and L1, 5F3 and L2, 5F3 and L3, 5F3 and L4, 5F3 and L5, 5F4 and L1, 5F4 and L2, 5F4 and L3, 5F4 and L4, or 5F4 and L5.

[0096] In one embodiment, the molecular scaffold may comprise at least one 3' flanking region and at least one loop motif region described in Tables 2 and 3. As a non-limiting example, the molecular scaffold can comprise 3F1 and L1, 3F1 and L2, 3F1 and L3, 3F1 and L4, 3F1 and L5, 3F2 and L1, 3F2 and L2, 3F2 and L3, 3F2 and L4, 3F2 and L5, 3F3 and L1, 3F3 and L2, 3F3 and L3, 3F3 and L4, 3F3 and L5, 3F4 and L1, 3F4 and L2, 3F4 and L3, 3F4 and L4, 3F4 and L5, 3F5 and L1, 3F5 and L2, 3F5 and L3, 3F5 and L4, 3F5 and L5, 3F6 and L1, 3F6 and L2, 3F6 and L3, 3F6 and L4 or 3F6 and L5.

[0097] In one embodiment, the molecular scaffold may comprise at least one 5' flanking region and at least a 3' flanking region as described in Tables 1 and 3. As a non-limiting example, the molecular scaffold may comprise 5F1 and 3F1, 5F1 and 3F2, 5F1 and 3F3, 5F1 and 3F4, 5F1 and 3F5, 5F1 and 3F6, 5F2 and 3F1, 5F2 and 3F2, 5F2 and 3F3, 5F2 and 3F4, 5F2 and 3F5, 5F2 and 3F6, 5F3 and 3F1, 5F3 and 3F2, 5F3 and 3F3, 5F3 and 3F4, 5F3 and 3F5, 5F3 and 3F6, 5F4 and 3F1, 5F4 and 3F2, 5F4 and 3F3, 5F4 and 3F4, 5F4 and 3F5, 5F4 and 3F6.

[0098] In one embodiment, the molecular scaffold may comprise at least one 5' flanking region, at least one loop motif region and at least one 3' flanking region. As a non-limiting example, the molecular scaffold may comprise 5F1, L1 and 3F1; 5F1, L1 and 3F2; 5F1, L1 and 3F3; 5F1, L1 and 3F4; 5F1, L1 and 3F5; 5F1, L1 and 3F6; 5F2, L1 and 3F1; 5F2, L1 and 3F2; 5F2, L1 and 3F3; 5F2, L1 and 3F4; 5F2, L1 and 3F5; 5F2, L1 and 3F6; 5F3, L1 and 3F1; 5F3, L1 and 3F2; 5F3, L1 and 3F3; 5F3, L1 and 3F4; 5F3, L1 and 3F5; 5F3, L1 and 3F6; 5F4, L1 and 3F1; 5F4, L1 and 3F3; 1 and 3F2; 5F4, L1 and 3F3; 5F4, L1 and 3F4; 5F4, L1 and 3F5; 5F4, L1 and 3F6; 5F1, L2 and 3F1; 5F1, L2 and 3F2; 5F1, L2 and 3F3; 5F1, L2 and 3F4; 5F1, L2 and 3F5; 5F1, L2 and 3F6; 5F2, L2 and 3F1; 5F2, L2 and 3F2; 5F2, L2 and 3F3; 5F2, L2 and 3F4; 5F2, L2 and 3F5; 5F2, L2 and 3F6; 5F3, L2 and 3F1; 5F3, L2 and 3F2; 5F3, L2 and 3F3; 5F3, L2 and 3F4; 5F3, L2 and 3F5; 5F3, L2, and 3F6; 5F4, L2, and 3F1; 5F4, L2, and 3F2; 5F4, L2, and 3F3; 5F4, L2, and 3F4; 5F4, L2, and 3F5; 5F4, L2, and 3F6; 5F1, L3, and 3F1; 5F1, L3, and 3F2; 5F1, L3, and 3F3; 5F1, L3, and 3F4; 5F1, L3, and 3F5; 5F1, L3, and 3F6; 5F2, L3, and 3F1; 5F2, L3, and 3F2; 5F2, L3, and 3F3; 5F2, L3, and 3F4; 5F2, L3, and 3F5; 5F2, L3, and 3F6; 5F3, L3, and 3F1; 5F3, L3, and 3F2; 5F3, L 3 and 3F3; 5F3, L3 and 3F4; 5F3, L3 and 3F5; 5F3, L3 and 3F6; 5F4, L3 and 3F1; 5F4, L3 and 3F2; 5F4, L3 and 3F3; 5F4, L3 and 3F4; 5F4, L3 and 3F5; 5F4, L3 and 3F6; 5F1, L4 and 3F1; 5F1, L4 and 3F2; 5F1, L4 and 3F3; 5F1, L4 and 3F4; 5F1, L4 and 3F5; 5F1, L4 and 3F6; 5F2, L4 and 3F1; 5F2, L4 and 3F2; 5F2, L4 and 3F3; 5F2, L4 and 3F4; 5F2, L4 and 3F5; 5F2, L4 and 3F6;5F3, L4 and 3F1; 5F3, L4 and 3F2; 5F3, L4 and 3F3; 5F3, L4 and 3F4; 5F3, L4 and 3F5; 5F3, L4 and 3F6; 5F4, L4 and 3F1; 5F4, L4 and 3F2; 5F4, L4 and 3F3; 5F4, L4 and 3F4; 5F4, L4 and 3F5; 5F4, L4 and 3F6; 5F1, L5 and 3F1; 5F1, L5 and 3F2; 5F1, L5 and 3F3; 5F1, L5 and 3F4; 5F1, L5 and 3F5; 5F1, L5 and 3F6; 5F2, L5 and 3F1; 5F2, L5 and 3F2; 5F2, L5 and 3F3; 5F2, L5 and 3F4; 5F2, L5 and 3F5; 5F2, L5 and 3F6; 5F3, L5 and 3F1; 5F3, L5 and 3F2; 5F3, L5 and 3F3; 5F3, L5 and 3F4; 5F3, L5 and 3F5; 5F3, L5 and 3F6; 5F4, L5 and 3F1; 5F4, L5 and 3F2; 5F4, L5 and 3F3; 5F4, L5 and 3F4; 5F4, L5 and 3F5; or 5F4, L5 and 3F6. ;

[0099] In one embodiment, the molecular scaffold may comprise one or more linkers known in the art. The linkers may separate the regions or separate one molecular scaffold from another. As a non-limiting example, the molecular scaffold may be polycistronic.

[0100] In one embodiment, at least one of the following properties is used to design regulatory polynucleotides: loop variants, seed mismatch / bulge / wobble variants, stem mismatch, loop variants and vassal stem mismatch variants, seed mismatch and base stem mismatch variants, stem mismatch and base stem mismatch variants, seed wobble and base stem wobble variants, or stem sequence variants.

[0101] In one embodiment, the molecular scaffold can be located downstream of a promoter (e.g., but not limited to, CMV, U6, CBA or CBA promoter with SV40 intron). In addition, the molecular scaffold can also be located upstream of a polyadenylation sequence. As a non-limiting example, the molecular scaffold can be located within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more than 30 nucleotides downstream of the promoter and / or upstream of the polyadenylation sequence. As another non-limiting example, the molecular scaffold can be located within 1-5, 1-10, 1-15, 1-20, 1-25, 1-30, 5-10, 5-15, 5-20, 5-25, 5-30, 10-15, 10-20, 10-25, 10-30, 15-20, 15-25, 15-30, 20-25, 20-30 or 25-30 nucleotides downstream of the promoter and / or upstream of the polyadenylation sequence. As a non-limiting example, the molecular scaffold can be located within the first 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25% or more than 25% of the nucleotides downstream of the promoter and / or upstream of the polyadenylation sequence. As a further non-limiting example, the molecular scaffold may be located within the first 1-5%, 1-10%, 1-15%, 1-20%, 1-25%, 5-10%, 5-15%, 5-20%, 5-25%, 10-15%, 10-20%, 10-25%, 15-20%, 15-25% or 20-25% downstream of the promoter and / or upstream of the polyadenylation sequence.

[0102] In one embodiment, the molecular scaffold can be located upstream of the polyadenylation sequence. In addition, the molecular scaffold can be located downstream of a promoter (e.g., but not limited to, CMV, U6, CBA or CBA promoter with SV40 intron). As a non-limiting example, the molecular scaffold can be located within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more than 30 nucleotides downstream of the promoter and / or upstream of the polyadenylation sequence. As another non-limiting example, the molecular scaffold can be located within 1-5, 1-10, 1-15, 1-20, 1-25, 1-30, 5-10, 5-15, 5-20, 5-25, 5-30, 10-15, 10-20, 10-25, 10-30, 15-20, 15-25, 15-30, 20-25, 20-30 or 25-30 nucleotides downstream of the promoter and / or upstream of the polyadenylation sequence. As a non-limiting example, the molecular scaffold can be located within the first 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25% or more than 25% of the nucleotides downstream of the promoter and / or upstream of the polyadenylation sequence. As a further non-limiting example, the molecular scaffold may be located within the first 1-5%, 1-10%, 1-15%, 1-20%, 1-25%, 5-10%, 5-15%, 5-20%, 5-25%, 10-15%, 10-20%, 10-25%, 15-20%, 15-25% or 20-25% downstream of the promoter and / or upstream of the polyadenylation sequence.

[0103] In one embodiment, the molecular scaffold may be located in a scAAV.

[0104] In one embodiment, the molecular scaffold may be located in ssAAV.

[0105] In one embodiment, the molecular scaffold may be located near the 5' end of the flip ITR. In another embodiment, the molecular scaffold may be located near the 3' end of the flip ITR. In another embodiment, the molecular scaffold may be located near the 5' end of the flop ITR. In another embodiment, the molecular scaffold may be located near the 3' end of the flop ITR. In one embodiment, the molecular scaffold may be located between the 5' end of the flip ITR and the 3' end of the flop ITR. In one embodiment, the molecular scaffold may be located between the 3' end of the flip ITR and the 5' end of the flip ITR (e.g., midway between the 5' end of the flip ITR and the 3' end of the flop ITR, or midway between the 3' end of the flop ITR and the 5' end of the flip ITR). As a non-limiting example, the molecular scaffold can be located within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more than 30 nucleotides downstream of the 5' or 3' end of the ITR (e.g., Flip or Flop ITR). As a non-limiting example, the molecular scaffold can be located within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more than 30 nucleotides upstream of the 5' or 3' end of the ITR (e.g., Flip or Flop ITR). As a further non-limiting example, the molecular scaffold can be located within 1-5, 1-10, 1-15, 1-20, 1-25, 1-30, 5-10, 5-15, 5-20, 5-25, 5-30, 10-15, 10-20, 10-25, 10-30, 15-20, 15-25, 15-30, 20-25, 20-30 or 25-30 nucleotides downstream of the 5' or 3' end of the ITR (e.g., Flip or Flop ITR). As a further non-limiting example, the molecular scaffold can be located within 1-5, 1-10, 1-15, 1-20, 1-25, 1-30, 5-10, 5-15, 5-20, 5-25, 5-30, 10-15, 10-20, 10-25, 10-30, 15-20, 15-25, 15-30, 20-25, 20-30 or 25-30 nucleotides upstream of the 5' or 3' end of the ITR (e.g., Flip or Flop ITR).As a non-limiting example, the molecular scaffold can be located within the first 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25% or more than 25% of the nucleotides upstream of the 5' or 3' end of the ITR (e.g., Flip or Flop ITR). As another non-limiting example, the molecular scaffold can be located within the first 1-5%, 1-10%, 1-15%, 1-20%, 1-25%, 5-10%, 5-15%, 5-20%, 5-25%, 10-15%, 10-20%, 10-25%, 15-20%, 15-25% or 20-25% of the 5' or 3' end of the ITR (e.g., Flip or Flop ITR).

[0106] Expression vector

[0107] In one embodiment, an expression vector (eg, an AAV vector) may comprise at least one regulatory polynucleotide comprising at least one molecular scaffold described herein.

[0108] In one embodiment, the expression vector may comprise, from ITR to ITR (from 5' to 3'), ITR, promoter, intron, regulatory polynucleotide, poly(A) sequence and ITR.

[0109] Genome size

[0110] In one embodiment, the vector genome comprising the nucleic acid sequence encoding the regulatory polynucleotide described herein can be a single-stranded or double-stranded vector genome. The size of the vector genome can be small, medium, large or maximum size. In addition, the vector genome can include a promoter and a poly(A) tail.

[0111] In one embodiment, the vector genome comprising the nucleic acid sequence encoding the regulatory polynucleotide described herein can be a small single-stranded vector genome. The small single-stranded vector genome can be 2.7-3.5kb in size, such as about 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4 and 3.5kb in size. As a non-limiting example, the small single-stranded vector genome can be 3.2kb in size. In addition, the vector genome can include a promoter and a polyadenylic acid tail.

[0112] In one embodiment, the vector genome comprising the nucleic acid sequence encoding the regulatory polynucleotide described herein can be a small double-stranded vector genome. The small double-stranded vector genome can be 1.3-1.7kb in size, such as about 1.3, 1.4, 1.5, 1.6 and 1.7kb in size. As a non-limiting example, the small double-stranded vector genome can be 1.6kb in size. In addition, the vector genome can include a promoter and a polyadenylic acid tail.

[0113] In one embodiment, the vector genome comprising the nucleic acid sequence encoding the regulatory polynucleotide described herein can be a medium single-stranded vector genome. The medium single-stranded vector genome can be 3.6-4.3kb in size, such as about 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2 and 4.3kb in size. As a non-limiting example, the medium single-stranded vector genome can be 4.0kb in size. In addition, the vector genome can include a promoter and a polyadenylic acid tail.

[0114] In one embodiment, the vector genome comprising the nucleic acid sequence encoding the regulatory polynucleotide described herein can be a medium double-stranded vector genome. The medium double-stranded vector genome can be 1.8-2.1kb in size, such as about 1.8, 1.9, 2.0 and 2.1kb in size. As a non-limiting example, the medium double-stranded vector genome can be 2.0kb in size. In addition, the vector genome can include a promoter and a polyadenylic acid tail.

[0115] In one embodiment, the vector genome comprising the nucleic acid sequence encoding the regulatory polynucleotide described herein can be a large single-stranded vector genome. Large single-stranded vector genome can be 4.4-6.0kb size, such as about 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 and 6.0kb size. As a non-limiting example, the large single-stranded vector genome can be 4.7kb size. As another non-limiting example, the large single-stranded vector genome can be 4.8kb size. As another non-limiting example, the large single-stranded vector genome can be 6.0kb size. In addition, the vector genome can include a promoter and a poly A tail.

[0116] In one embodiment, the vector genome comprising the nucleic acid sequence encoding the regulatory polynucleotide described herein can be a large double-stranded vector genome. The large double-stranded vector genome can be 2.2-3.0kb in size, such as about 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 and 3.0kb in size. As a non-limiting example, the large double-stranded vector genome can be 2.4kb in size. In addition, the vector genome can include a promoter and a polyadenylic acid tail.

[0117] Promoter

[0118] Those skilled in the art will recognize that target cells may require specific promoters, including, but not limited to, species-specific, inducible, tissue-specific, or cell cycle-specific promoters (Parr et al., Nat. Med. 3:1145-9 (1997); the contents of which are incorporated herein by reference in their entirety).

[0119] In one embodiment, the promoter is one that is considered to be effective for the payload in the regulatory polynucleotide.

[0120] In one embodiment, the promoter is one that is recognized to be effective in the targeted cell.

[0121] In one embodiment, the promoter is a weak promoter that provides for expression of the payload in a targeted tissue (e.g., but not limited to, nervous system tissue) for a period of time. Expression can last for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days ... weeks, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years or a period of time exceeding 10 years. Expression can last for 1-5 hours, 1-12 hours, 1-2 days, 1-5 days, 1-2 weeks, 1-3 weeks, 1-4 weeks, 1-2 months, 1-4 months, 1-6 months, 2-6 months, 3-6 months, 3-9 months, 4-8 months, 6-12 months, 1-2 years, 1-5 years, 2-5 years, 3-6 years, 3-8 years, 4-8 years, or 5-10 years. As a non-limiting example, the promoter is a weak promoter for sustained expression of the payload in neural tissue.

[0122] In one embodiment, the promoter may be a promoter of less than 1 kb. The promoter may have a size of 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 50 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, or a length exceeding 800. The promoter may have a length of between 200-300, 200-400, 200-500, 200-600, 200-700, 200-800, 300-400, 300-500, 300-600, 300-700, 300-800, 400-500, 400-600, 400-700, 400-800, 500-600, 500-700, 500-800, 600-700, 600-800, or 700-800.

[0123] In one embodiment, the promoter can be a combination of two or more components (for example, but not limited to, CMV and CBA). Each component can have 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 400, 410, 420, 430, 440, 450, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800 or a length exceeding 800. Each component can have a length of between 200-300, 200-400, 200-500, 200-600, 200-700, 200-800, 300-400, 300-500, 300-600, 300-700, 300-800, 400-500, 400-600, 400-700, 400-800, 500-600, 500-700, 500-800, 600-700, 600-800, or 700-800. As a non-limiting example, the promoter is a combination of a 382-nucleotide CMV-enhancer sequence and a 260-nucleotide CBA-promoter sequence.

[0124] In one embodiment, the vector genome comprises at least one element to enhance transgenic target specificity and expression (see, e.g., Powell et al. Viral Expression Cassette Elements to Enhance Transgene Target Specificity and Expression in Gene Therapy, 2015; the contents of which are incorporated herein by reference in their entirety). Non-limiting examples of elements for enhancing transgenic target specificity and expression include promoters, endogenous miRNAs, post-transcriptional regulatory elements (PREs), polyadenylation (PolyA) signal sequences and upstream enhancers (USEs), CMV enhancers, and introns.

[0125] In one embodiment, the vector genome comprises at least one element to enhance transgene target specificity and expression (see, e.g., Powell et al. Viral Expression Cassette Elements to Enhance Transgene Target Specificity and Expression in Gene Therapy, 2015; the contents of which are incorporated herein by reference in their entirety), such as a promoter. Promoters that promote expression in most tissues include, but are not limited to, human elongation factor 1α-subunit (EF1α), immediate early cytomegalovirus (CMV), chicken β-actin (CBA) and its derivatives CAG, β-glucuronidase (GUSB) or ubiquitin C (UBC). Tissue-specific expression elements can be used to restrict expression to specific cell types, for example, but not limited to, a nervous system promoter that can be used to restrict expression to neurons, astrocytes or oligodendrocytes. Non-limiting examples of tissue-specific expression elements for neurons include neuron-specific enolase (NSE), platelet-derived growth factor (PDGF), platelet-derived growth factor B-chain (PDGF-β), synaptophysin (Syn), methyl-CpG binding protein 2 (MeCP2), CaMKII, mGluR2, NFL, NFH, nβ2, PPE, Enk, and EAAT2 promoters. A non-limiting example of tissue-specific expression elements for astrocytes includes glial fibrillary acid protein (GFAP) and EAAT2 promoters. A non-limiting example of tissue-specific expression elements for oligodendrocytes is the myelin basic protein (MBP) promoter.

[0126] In one embodiment, the vector genome comprises a ubiquitous promoter. Non-limiting examples of ubiquitous promoters include CMV, CBA (including derivatives CAG, CBh, etc.), EF-1α, PGK, UBC, GUSB (hGBp) and UCOE (promoter of HNRPA2B1-CBX3). Yu et al. (Molecular Pain 2011, 7:63; the contents of which are incorporated herein by reference in their entirety) evaluated the expression of eGFP under CAG, EFIα, PGK and UBC promoters in rat DRG cells and primary DRG cells using lentiviral vectors, and found that UBC showed weaker expression than the other three promoters, and there was only 10-12% glial expression for all promoters. Soderblom et al. (E. Neuro 2015; the contents of which are incorporated herein by reference in their entirety) evaluated the expression of eGFP in AAV8 with CMV and UBC promoters and AAV2 with CMV promoter after injection into the motor cortex. Intranasal administration of plasmids containing the UBC or EFIα promoters exhibited sustained airway expression greater than expression with the CMV promoter (see, e.g., Gill et al., Gene Therapy 2001, Vol. 8, 1539-1546; the contents of which are incorporated herein by reference in their entirety). Husain et al. (Gene Therapy 2009; the contents of which are incorporated herein by reference in their entirety) evaluated HβH constructs with the hGUSB promoter, the HSV-1 LAT promoter, and the NSE promoter, and found that the HβH constructs exhibited weaker expression than NSE in the mouse brain. Passini and Wolfe (J. Virol. 2001, 12382-12392, the contents of which are incorporated herein by reference in their entirety) evaluated the long-term effects of HβH vectors after intraventricular injection in neonatal mice and found that there was persistent expression for at least 1 year. When using the NF-L and NF-H promoters to compare with CMV-lacZ, CMV-luc, EF, GFAP, hENK, nAChR, PPE, PPE+wpre, NSE(0.3kb), NSE(1.8kb), and NSE(1.8kb+wpre), Xu et al. (Gene Therapy 2001, 8, 1323-1332; the contents of which are incorporated herein by reference in their entirety) found low expression in all brain regions. Xu et al. found that the promoter activity was in descending order of NSE(1.8kb), EF, NSE(0.3kb), GFAP, CMV, hENK, PPE, NFL, and NFH. NFL is a 650 nucleotide promoter and NFH is a 920 nucleotide promoter, neither of which is present in the liver, but NFH is abundant in sensory proprioceptive neurons, brain, and spinal cord, and NFH is present in the heart.Scn8a is a 470 nucleotide promoter expressed ubiquitously in DRG, spinal cord, and brain, with particularly high expression seen in hippocampal neurons and cerebellar Purkinje cells, cortex, thalamus, and hypothalamus (see, e.g., Drews et al. 2007 and Raymond et al. 2004; the contents of each of which are incorporated herein by reference in their entirety).

[0127] In one embodiment, the vector genome comprises a UBC promoter. The UBC promoter may have a size of 300-350 nucleotides. As a non-limiting example, the UBC promoter is 332 nucleotides.

[0128] In one embodiment, the vector genome comprises a GUSB promoter. The GUSB promoter may have a size of 350-400 nucleotides. As a non-limiting example, the GUSB promoter is 378 nucleotides. As a non-limiting example, the construct may be AAV-promoter-CMV / globin intron-hFXN-RBG, wherein the AAV may be self-complementary, and the AAV may be a DJ serotype.

[0129] In one embodiment, the vector genome comprises an NFL promoter. The NFL promoter may have a size of 600-700 nucleotides. As a non-limiting example, the NFL promoter is 650 nucleotides. As a non-limiting example, the construct may be AAV-promoter-CMV / globin intron-hFXN-RBG, wherein the AAV may be self-complementary, and the AAV may be a DJ serotype.

[0130] In one embodiment, the vector genome comprises a NFH promoter. The NFH promoter may have a size of 900-950 nucleotides. As a non-limiting example, the NFH promoter is 920 nucleotides. As a non-limiting example, the construct may be AAV-promoter-CMV / globin intron-hFXN-RBG, wherein the AAV may be self-complementary, and the AAV may be a DJ serotype.

[0131] In one embodiment, the vector genome comprises a scn8a promoter. The scn8a promoter may have a size of 450-500 nucleotides. As a non-limiting example, the scn8a promoter is 470 nucleotides. As a non-limiting example, the construct may be AAV-promoter-CMV / globin intron-hFXN-RBG, wherein the AAV may be self-complementary, and the AAV may be a DJ serotype.

[0132] In one embodiment, the vector genome comprises a FXN promoter.

[0133] In one embodiment, the vector genome comprises a PGK promoter.

[0134] In one embodiment, the vector genome comprises a CBA promoter.

[0135] In one embodiment, the vector genome comprises a CMV promoter.

[0136] In one embodiment, the vector genome comprises a liver or skeletal muscle promoter. Non-limiting examples of liver promoters include hAAT and TBG. Non-limiting examples of skeletal muscle promoters include desmin, MCK, and C5-12.

[0137] In one embodiment, the expression vector comprises an enhancer element, a promoter and / or a 5'UTR intron. The enhancer may be, but is not limited to, a CMV enhancer, the promoter may be, but is not limited to, CMV, CBA, UBC, GUSB, NSE, Sunapsin, MeCP2 and GFAP promoters, and the 5'UTR / intron may be, but is not limited to, SV40 and CBA-MVM. As a non-limiting example, the enhancer, promoter and / or intron used in combination can be: (1) CMV enhancer, CMV promoter, SV40 5'UTR intron; (2) CMV enhancer, CBA promoter, SV 40 5'UTR intron; (3) CMV enhancer, CBA promoter, CBA-MVM 5'UTR intron; (4) UBC promoter; (5) GUSB promoter; (6) NSE promoter; (7) synapsin promoter; (8) MeCP2 promoter and (9) GFAP promoter.

[0138] In one embodiment, the expression vector has an engineered promoter.

[0139] Intron

[0140] In one embodiment, the vector genome comprises at least one element to enhance transgene target specificity and expression (see, e.g., Powell et al. Viral Expression Cassette Elements to Enhance Transgene Target Specificity and Expression in Gene Therapy, 2015; the contents of which are incorporated herein by reference in their entirety), such as an intron. Non-limiting examples of introns include MVM (67-97 bp), F.IX truncated intron 1 (300 bp), β-globin SD / immunoglobulin heavy chain splice acceptor (250 bp), adenovirus splice donor / immunoglobulin splice acceptor (500 bp), SV40 late splice donor / splice acceptor (19S / 16S) (180 bp), and hybrid adenovirus splice donor / IgG splice acceptor (230 bp).

[0141] In one embodiment, the intron can be 100-500 nucleotides in length. The intron can have a length of 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490 or 500. The promoter may have a length of between 80-100, 80-120, 80-140, 80-160, 80-180, 80-200, 80-250, 80-300, 80-350, 80-400, 80-450, 80-500, 200-300, 200-400, 200-500, 300-400, 300-500, or 400-500.

[0142] Introduced into cells

[0143] The regulatory polynucleotides of the present invention can be introduced into host cells using any of a variety of protocols. Infection can be achieved using a viral vector comprising the regulatory polynucleotide. Examples of suitable viral vectors include replication-defective retroviral vectors, adenoviral vectors, adeno-associated vectors, and lentiviral vectors.

[0144] According to the present invention, viral vectors for use in therapeutic and / or diagnostic agents comprise viruses that have been simplified or reduced to the minimum components necessary for transduction of a target nucleic acid payload or cargo.

[0145] In this way, viral vectors are engineered as vehicles for specific delivery while lacking the deleterious replication and / or integration features found in wild-type viruses.

[0146] As used herein, "vector" is any molecule or part of a vector that transports, transduces, or otherwise acts as a heterologous molecule (such as a polynucleotide of the present invention) or a regulatory polynucleotide of the present invention. "Viral vector" is a vector comprising one or more polynucleotide regions that encode or contain a target payload molecule, e.g., a transgenic, a polynucleotide encoding one or more polypeptides, or a regulatory nucleic acid. The viral vector of the present invention can be produced recombinantly and can be based on an adeno-associated virus (AAV) parent or reference sequence. Serotypes that may be useful in the present invention include any of those produced from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9 (hu14), AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAV-DJ, and AAV-DJ8.

[0147] In one embodiment, the serotype useful in the present invention may be AAV-DJ8. The amino acid sequence of AAV-DJ8 may comprise two or more mutations to remove the heparin binding domain (HBD). As a non-limiting example, the AAV-DJ sequence described as SEQ ID NO: 1 in U.S. Pat. No. 7,588,772 (the contents of which are incorporated herein by reference in their entirety) may comprise two mutations: (1) R587Q, wherein the arginine (R; Arg) at amino acid 587 is changed to glutamine (Q; Gln) and (2) R590T, wherein the arginine (R; Arg) at amino acid 590 is changed to threonine (T; Thr). As another non-limiting example, three mutations may be included: (1) K406R, in which the lysine (K; Lys) at amino acid 406 is changed to arginine (R; Arg), (2) R587Q, in which the arginine (R; Arg) at amino acid 587 is changed to glutamine (Q; Gln), and (3) R590T, in which the arginine (R; Arg) at amino acid 590 is changed to threonine (T; Thr).

[0148] AAV vectors can also include self-complementary AAV vectors (scAAV). scAAV vectors contain two DNA strands that mate together to form double-stranded DNA. By skipping second strand synthesis, scAAV allows rapid expression in cells.

[0149] In one embodiment, the AAV vector used in the present invention is scAAV.

[0150] In one embodiment, the regulatory polynucleotide may be introduced into cells from any relevant species (eg, but not limited to, human, dog, mouse, rat, or monkey).

[0151] In one embodiment, the regulatory polynucleotides may be introduced into cells associated with the disease to be treated. As a non-limiting example, the disease is ALS and the target cells are motor neurons and astrocytes.

[0152] In one embodiment, the regulatory polynucleotide may be introduced into cells that have high endogenous expression levels of the target sequence.

[0153] In another embodiment, the regulatory polynucleotide may be introduced into cells that have low endogenous expression levels of the target sequence.

[0154] In one embodiment, the cells may be those with high AAV transduction efficiency.

[0155] In one embodiment, cells that can be used for in vitro analysis of regulatory polynucleotides include, but are not limited to, HEK293, HeLa, human primary astrocytes, a human astrocyte cell line (U251MG), SH-SY5Y-neurons, and human iPSC-derived motor neuron progenitor cells.

[0156] Target nucleic acid

[0157] The regulatory polynucleotides of the present invention can target any gene or nucleic acid construct, including coding and non-coding genes. Genes (DNA or mRNA) encoding human or primate proteins can be targeted. In addition, non-coding genes, such as long non-coding RNA (lncRNA), can also be targeted.

[0158] Examples of such lncRNA molecules and RNAi constructs designed to target such lncRNAs are taught in International Publication No. WO2012 / 018881A2, the contents of which are incorporated herein by reference in their entirety, any of which may be targeted by or encoded by the regulatory polynucleotide, respectively.

[0159] In one embodiment, the regulatory polynucleotides of the present invention can target any gene known in the art. As a non-limiting example, the gene can be SOD1.

[0160] In one embodiment, the regulatory polynucleotide can target a sequence of 15-19 nucleotides in length. As a non-limiting example, the target can be any sequence described in Table 1. As another non-limiting example, the target can be nucleotides 406-424 of NM_000454.4. As another non-limiting example, the target can be nucleotides 645-661 of NM_000454.4.

[0161] In one embodiment, the regulatory polynucleotide can target a sequence of 21 nucleotides in length. In one aspect, the target can be any 21-mer sequence of NM_000454.4 or any gene known in the art. As a non-limiting example, the target can be nucleotides 521-541 of NM_000454.4. As another non-limiting example, the target can be nucleotides 639-659 of NM_000454.4. As another non-limiting example, the target can be nucleotides 640-660 of NM_000454.4. As another non-limiting example, the target can be nucleotides 645-665 of NM_000454.4. As another non-limiting example, the target can be nucleotides 664-684 of NM_000454.4.

[0162] In one embodiment, the regulatory polynucleotides can be designed to target any gene or mRNA in the human genome, for example, genes associated with CNS disorders (eg, but not limited to, Huntington's disease, ALS, etc.).

[0163] Pharmaceutical composition

[0164] Although the descriptions of pharmaceutical compositions provided herein, such as those regulatory polynucleotides (including encoding plasmids or expression vectors, such as viruses, e.g., AAV) comprising a payload to be delivered, are primarily directed to pharmaceutical compositions suitable for administration to humans, the skilled artisan will appreciate that such compositions are generally suitable for administration to any other animal, e.g., non-human animals, such as non-human mammals. Modifications to pharmaceutical compositions suitable for administration to humans so as to make the compositions suitable for administration to a variety of animals are well understood, and a veterinary pharmacologist of ordinary skill can design and / or perform such modifications with only ordinary (if any) experimentation. Subjects to whom the pharmaceutical compositions are contemplated for administration include, but are not limited to, humans and / or other primates; mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and / or rats; and / or birds, including commercially relevant birds such as poultry, chickens, ducks, geese, and / or turkeys.

[0165] In certain embodiments, the composition is administered to a human, human patient, or subject.For the purposes of this disclosure, the phrase "active ingredient" generally refers to a viral vector carrying the payload or a regulatory polynucleotide payload molecule delivered with a viral vector described herein.

[0166] The formulations of the pharmaceutical compositions described herein can be prepared by any method known in the art of pharmacology or developed thereafter. In general, such preparation methods include the steps of combining the active ingredient with an excipient and / or one or more other adjuvants, and then, if necessary and / or desirable, packaging, shaping and / or packaging the product into desired single or multiple dose units.

[0167] The relative amounts of active ingredient, pharmaceutically acceptable excipient and / or any additional ingredients in a pharmaceutical composition according to the invention will vary, depending on the identity, size and / or condition of the subject being treated, and further on the route by which the composition is to be administered.

[0168] preparation

[0169] The regulatory polynucleotides or viral vectors encoding them can be formulated with one or more excipients to: (1) increase stability; (2) increase cell transfection or transduction; (3) allow for sustained or delayed release; or (4) alter biodistribution (e.g., to target the viral vector to a specific tissue or cell type).

[0170] The formulations of the present invention may include, but are not limited to, saline, lipid-like substances, liposomes, lipid nanoparticles, polymers, lipid complexes, core-shell nanoparticles, peptides, proteins, cells transfected with viral vectors (e.g., for transplantation into a subject), nanoparticle mimics, and combinations thereof. In addition, the viral vectors of the present invention may be formulated using self-assembling nucleic acid nanoparticles.

[0171] The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparation methods include the steps of combining the active ingredient with an excipient and / or one or more other auxiliary agents.

[0172] Pharmaceutical compositions according to the present disclosure can be prepared, packaged and / or sold in bulk, as a single unit dose and / or as multiple single unit doses. "Unit dose" as used herein means a discrete amount of a pharmaceutical composition comprising a predetermined amount of active ingredient. The amount of active ingredient is generally equal to the dosage of the active ingredient to be administered to the subject, and / or a suitable fraction of such dosage, such as one-half or one-third of such dosage.

[0173] The relative amounts of the active ingredient, pharmaceutically acceptable excipients and / or any additional ingredients in a pharmaceutical composition according to the present disclosure may vary, depending on the nature, size and / or condition of the subject being treated, and also on the route by which the composition is to be administered. For example, the composition may contain 0.1% to 99% (w / w) of the active ingredient. As an example, the composition may contain 0.1% to 100%, e.g., 0.5 to 50%, 1-30%, 5-80%, at least 80% (w / w) of the active ingredient.

[0174] In certain embodiments, the formulations described herein may contain at least one payload molecule. As non-limiting examples, the formulation may contain 1, 2, 3, 4 or 5 regulatory polynucleotide payload molecules. In one embodiment, the formulation may contain a regulatory polynucleotide payload construct, which targets a protein selected from, for example, but not limited to, human proteins, veterinary proteins, bacterial proteins, biological proteins, antibodies, immunogenic proteins, therapeutic peptides and proteins, secreted proteins, plasma membrane proteins, cytoplasmic and cytoskeletal proteins, intracellular membrane-bound proteins, nucleoproteins, proteins associated with human diseases and / or proteins associated with non-human diseases. In one embodiment, the formulation contains a payload construct of at least 3 targeted proteins.

[0175] In certain embodiments, pharmaceutically acceptable excipients can be at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% pure. In certain embodiments, excipients are approved for the human and for veterinary applications. In certain embodiments, excipients can be approved by the U.S. Food and Drug Administration. In certain embodiments, excipients can belong to pharmaceutical grade. In certain embodiments, excipients can meet the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia and / or the International Pharmacopoeia.

[0176] Excipients used herein include, but are not limited to, any and all of solvents, dispersion media, diluents or other liquid vehicles, dispersion or suspension aids, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, etc., only if they are suitable for the desired specific dosage form. Various excipients for preparing pharmaceutical compositions and techniques for preparing compositions are known in the art (see Remington: The Science and Practice of Pharmacy, 21st edition, A.R. Gennaro, Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference in their entirety). The use of conventional excipient media can be foreseen within the scope of the present disclosure, but except that any conventional excipient media may be incompatible with the substance or its derivatives, such as by producing any undesirable biological effects or otherwise interacting with any other components of the pharmaceutical composition in a harmful manner.

[0177] Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dried starch, corn starch, powdered sugar, etc., and / or combinations thereof.

[0178] No active ingredients

[0179] In certain embodiments, the regulatory polynucleotide formulation may include at least one excipient, which is an inactive ingredient. The term "inactive ingredient" as used herein means one or more inactive agents included in the formulation. In certain embodiments, all, none or some of the inactive ingredients that can be used in the formulation of the present invention can be approved by the U.S. Food and Drug Administration (FDA).

[0180] The preparation of the viral vector carrying the regulatory polynucleotide disclosed herein may include cations or anions. In one embodiment, the preparation includes metal cations, such as, but not limited to, Zn2+, Ca2+, Cu2+, Mg+ and combinations thereof. As a non-limiting example, the preparation may include a polymer and a regulatory polynucleotide that forms a complex with a metal cation (see, e.g., U.S. Patent Nos. 6,265,389 and 6,555,525, each of which is incorporated herein by reference in its entirety).

[0181] Application

[0182] The viral vectors comprising the regulatory polynucleotides of the invention may be administered by any route that produces a therapeutically effective result. These include, but are not limited to, enteral (into the intestine), enteral, epidural (into the dura mater), oral (through the mouth), transdermal, epidural, intracerebral (into the brain), intraventricular (into the ventricles), intradermal (applied on the skin), intradermal (into the skin itself), subcutaneous (under the skin), nasal administration (through the nose), intravenous (into a vein), intravenous bolus, intravenous drip, intraarterial (into an artery), intramuscular (into a muscle), intracardiac (into the heart), intraosseous infusion (into the bone marrow), intrathecal (into the spinal canal), intraperitoneal (infusion or injection into the peritoneum), intravesical infusion, intravitreal (through the eye), intracavernous injection (into a pathological cavity), intracavitary (into the base of the penis) , intravaginal, intrauterine, extraamniotic, transdermal (diffusion through intact skin for systemic distribution), transmucosal (diffusion through mucous membranes), vaginal, insufflation (sniffing), sublingual, sublabial, enema, eye drops (on the conjunctiva), ear drops, otic (in or through the ear), buccal (superior to the cheek), conjunctival, dermal, dental (to one or more teeth), electroosmotic, intracervical, endosinusial, intratracheal, extracorporeal, hemodialysis, infiltration, interstitial, intraabdominal, intraamniotic, intraarticular, intrabiliary, intrabronchial, intracystic, intracartilaginous (in the cartilage), intracaudal (in the cauda equina), intracisternal (in the cerebellomedullary cistern), intracorneal (in the cornea), dental intracorneal, intracoronary (in the coronary artery), intracorporal (in the cavernosum (in the expandable space of the corpus cavernosum of the penis), intradiscal (in the intervertebral disc), intraductal (in the duct of a gland), intraduodenal (in the duodenum), intradural (in or below the dura mater), intraepidermal (to the epidermis), intraesophageal (to the esophagus), intragastric (in the stomach), intragingival (in the gums), intraileal (in the distal portion of the small intestine), intralesional (in a local lesion or introduced directly into a local lesion), intraluminal (in the lumen), intralymphatic (in the lymph), intramedullary (in the bone marrow cavity of a bone), intrameningeal (in the meninges), intraocular (in the eye), intraovarian (in the ovary intraspinal (in the spine), intrasynovial (in the synovial cavity of a joint), intratendinous (in a tendon), intratesticular (in a testis), intrathecal (in the cerebrospinal fluid at any level of the cerebrospinal axis), intrathoracic (in the chest cavity), intratubular (in a tube of an organ), intratumoral (in a tumor), intratympanic (in the aurus medium), intravascular (in one or more blood vessels), ventricular (in the heart ventricle), iontophoresis (with the aid of an electric current,In some embodiments, the compositions may be administered in a manner that allows them to cross the blood-brain barrier, vascular barrier, or other epithelial barrier. In one embodiment, the formulation for the route of administration may include at least one inactive ingredient.

[0183] Dosing

[0184] The present invention provides methods comprising administering to a subject in need thereof a viral vector according to the present invention and their regulatory polynucleotide payload or complex. A viral vector pharmaceutical composition, an imaging composition thereof, a diagnostic composition, or a preventive composition may be administered to a subject using any amount and any route of administration that is effective for preventing, treating, diagnosing, or imaging a disease, disorder, and / or condition (e.g., a disease, disorder, and / or condition associated with working memory loss). The exact amount required will vary between subjects, depending on the species, age, and overall condition of the subject, the severity of the disease, the specific composition, its mode of administration, its mode of activity, and the like. Compositions according to the present invention are typically formulated in unit dosage forms that are easy to administer and uniform in dosage. However, it should be understood that the total daily dosage of the composition of the present invention may be determined by the attending physician within the scope of reasonable medical judgment. The specific therapeutically effective, prophylactically effective, or appropriate imaging dosage level for any particular patient will depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, weight, general health, sex, and diet of the patient; the time of administration, route of administration, and excretion rate of the specific regulatory polynucleotide payload employed; the duration of the treatment; drugs used in combination or concomitantly with the specific compound employed; and like factors well known in the medical arts.

[0185] In certain embodiments, the dosage may be sufficient to deliver about 0.0001 mg / kg to about 100 mg / kg, about 0.001 mg / kg to about 0.05 mg / kg, about 0.005 mg / kg to about 0.05 mg / kg, about 0.001 mg / kg to about 0.005 mg / kg, about 0.05 mg / kg to about 0.5 mg / kg, about 0.01 mg / kg to about 50 mg / kg, about 0.1 mg / kg to about 40 mg / kg, about 0.5 mg / kg to about 100 mg / kg per day. The dosage level of the regulatory polynucleotide of kg to about 30mg / kg, about 0.01mg / kg to about 10mg / kg, about 0.1mg / kg to about 10mg / kg or about 1mg / kg to about 25mg / kg of the subject's body weight (once or more per day) is applied according to the viral vector pharmaceutical composition of the present invention, to obtain the desired treatment, diagnosis, prevention or imaging effect (see, for example, the range of unit doses described in International Publication No. WO2013078199, which is incorporated herein by reference in its entirety). The desired regulatory polynucleotide dosage can be delivered more than once (for example, more than once in a day). In certain embodiments, using multiple administrations (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 times or more administrations), the desired regulatory polynucleotide dosage can be delivered. When multiple administrations are adopted, a divided quantitative administration scheme, such as those described herein, can be used. "Divided dose" used herein is to divide a single unit dose or a total daily dose into 2 or more dosages, for example, twice or more administrations of a single unit dose. "Single unit dose" used herein is the dosage of any regulatory polynucleotide therapeutic agent used in one dose / at one time / single approach / single contact point (i.e., a single administration event). "Total daily dose" used herein is the amount given or prescribed in a 24-hour period. It can be administered as a single unit dose. In one embodiment, the viral vector comprising the regulatory polynucleotide of the present invention is administered to the subject in divided doses. They can be prepared in only buffer or in the preparation described herein.

[0186] In one embodiment, delivery of a composition according to the invention to a cell comprises a delivery rate defined by [VG / hour=mL / hour*VG / mL], where VG is the viral genome, VG / mL is the composition concentration, and mL / hour is the rate of extended delivery.

[0187] In one embodiment, delivery of a composition according to the present invention to a cell may comprise about 1 x 10 6 VG to about 1x10 16 Total concentration of VG / subject. In certain embodiments, delivery may comprise about 1x10 6 , 2x106 、3x10 6 、4x10 6 、5x10 6 、6x10 6 、7x10 6 、8x10 6 、9x10 6 、1x10 7 、2x10 7 、3x10 7 、4x10 7 、5x10 7 、6x10 7 、7x10 7 、8x10 7 、9x10 7 、1x10 8 、2x10 8 、3x10 8 、4x10 8 、5x10 8 、6x10 8 、7x10 8 、8x10 8 、9x10 8 、1x10 9 、2x10 9 、3x10 9 、4x10 9 、5x10 9 、6x10 9 、7x10 9 、8x10 9 、9x10 9 、1x10 10 、2x10 10 、3x10 10 、4x10 10 、5x10 10 、6x10 10 、7x10 10 、8x10 10 、9x10 10 、1x10 11 、2x10 11 、2.1x10 11 、2.2x10 11 、2.3x10 11 、2.4x10 11 、2.5x10 11 、2.6x10 11 、2.7x10 11 、2.8x10 11 、2.9x10 11、3x10 11 、4x10 11 、5x10 11 、6x10 11 、7x10 11 、7.1x10 11 、7.2x10 11 、7.3x10 11 、7.4x10 11 、7.5x10 11 、7.6x10 11 、7.7x10 11 、7.8x10 11 、7.9x10 11 、8x10 11 、9x10 11 、1x10 12 、1.1x10 12 、1.2x10 12 、1.3x10 12 、1.4x10 12 、1.5x10 12 、1.6x10 12 、1.7x10 12 、1.8x10 12 、1.9x10 12 、2x10 12 、3x10 12 、4x10 12 、4.1x10 12 、4.2x10 12 、4.3x10 12 、4.4x10 12 、4.5x10 12 、4.6x10 12 、4.7x10 12 、4.8x10 12 、4.9x10 12 、5x10 12 、6x10 12 、7x10 12 、8x10 12 、8.1x10 12 、8.2x10 12 、8.3x10 12 、8.4x10 12 、8.5x10 12 、8.6x10 12 、8.7x10 12 、8.8x10 12 、8.9x10 12 、9x1012 , 1x10 13 , 2x10 13 , 3x10 13 , 4x10 13 , 5x10 13 , 6x10 13 , 6.7x10 13 , 7x10 13 , 8x10 13 , 9x10 13 , 1x10 14 , 2x10 14 , 3x10 14 , 4x10 14 , 5x10 14 , 6x10 14 , 7x10 14 , 8x10 14 , 9x10 14 , 1x10 15 , 2x10 15 , 3x10 15 , 4x10 15 , 5x10 15 , 6x10 15 , 7x10 15 , 8x10 15 , 9x10 15 or 1x10 16 Composition concentration of VG / subject.

[0188] In one embodiment, delivery of a composition according to the present invention to cells may comprise about 1 x 10 6 VG / kg is about 1x10 16 In some embodiments, the delivery may contain about 1x10 6 , 2x10 6 , 3x10 6 , 4x10 6 , 5x10 6 , 6x10 6 , 7x10 6 , 8x10 6 , 9x10 6 , 1x10 7 , 2x10 7 , 3x10 7 , 4x10 7 , 5x10 7 , 6x10 7 , 7x10 7 , 8x10 7 , 9x10 7、1x10 8 、2x10 8 、3x10 8 、4x10 8 、5x10 8 、6x10 8 、7x10 8 、8x10 8 、9x10 8 、1x10 9 、2x10 9 、3x10 9 、4x10 9 、5x10 9 、6x10 9 、7x10 9 、8x10 9 、9x10 9 、1x10 10 、2x10 10 、3x10 10 、4x10 10 、5x10 10 、6x10 10 、7x10 10 、8x10 10 、9x10 10 、1x10 11 、2x10 11 、2.1x10 11 、2.2x10 11 、2.3x10 11 、2.4x10 11 、2.5x10 11 、2.6x10 11 、2.7x10 11 、2.8x10 11 、2.9x10 11 、3x10 11 、4x10 11 、5x10 11 、6x10 11 、7x10 11 、7.1x10 11 、7.2x10 11 、7.3x10 11 、7.4x10 11 、7.5x10 11 、7.6x10 11 、7.7x10 11 、7.8x10 11 、7.9x10 11 、8x10 11 、9x1011 、1x10 12 、1.1x10 12 、1.2x10 12 、1.3x10 12 、1.4x10 12 、1.5x10 12 、1.6x10 12 、1.7x10 12 、1.8x10 12 、1.9x10 12 、2x10 12 、3x10 12 、4x10 12 、4.1x10 12 、4.2x10 12 、4.3x10 12 、4.4x10 12 、4.5x10 12 、4.6x10 12 、4.7x10 12 、4.8x10 12 、4.9x10 12 、5x10 12 、6x10 12 、7x10 12 、8x10 12 、8.1x10 12 、8.2x10 12 、8.3x10 12 、8.4x10 12 、8.5x10 12 、8.6x10 12 、8.7x10 12 、8.8x10 12 、8.9x10 12 、9x10 12 、1x10 13 、2x10 13 、3x10 13 、4x10 13 、5x10 13 、6x10 13 、6.7x10 13 、7x10 13 、8x10 13 、9x10 13 、1x10 14 、2x10 14 、3x10 14 、4x10 14 、5x10 14 、6x10 14, 7x10 14 , 8x10 14 , 9x10 14 , 1x10 15 , 2x10 15 , 3x10 15 , 4x10 15 , 5x10 15 , 6x10 15 , 7x10 15 , 8x10 15 , 9x10 15 or 1x10 16 The concentration of the composition is VG / kg.

[0189] In one embodiment, each dose may be administered about 10 5 -10 6 Viral genome (unit).

[0190] In one embodiment, delivery of a composition according to the present invention to a cell may comprise about 1 x 10 6 VG / mL is about 1x10 16 In some embodiments, the delivery may contain about 1x10 6 , 2x10 6 , 3x10 6 , 4x10 6 , 5x10 6 , 6x10 6 , 7x10 6 , 8x10 6 , 9x10 6 , 1x10 7 , 2x10 7 , 3x10 7 , 4x10 7 , 5x10 7 , 6x10 7 , 7x10 7 , 8x10 7 , 9x10 7 , 1x10 8 , 2x10 8 , 3x10 8 , 4x10 8 , 5x10 8 , 6x10 8 , 7x10 8 , 8x10 8 , 9x10 8 , 1x10 9 , 2x10 9 , 3x10 9 , 4x109 、5x10 9 、6x10 9 、7x10 9 、8x10 9 、9x10 9 、1x10 10 、2x10 10 、3x10 10 、4x10 10 、5x10 10 、6x10 10 、7x10 10 、8x10 10 、9x10 10 、1x10 11 、2x10 11 、3x10 11 、4x10 11 、5x10 11 、6x10 11 、7x10 11 、8x10 11 、9x10 11 、1x10 12 、1.1x10 12 、1.2x10 12 、1.3x10 12 、1.4x10 12 、1.5x10 12 、1.6x10 12 、1.7x10 12 、1.8x10 12 、1.9x10 12 、2x10 12 、2.1x10 12 、2.2x10 12 、2.3x10 12 、2.4x10 12 、2.5x10 12 、2.6x10 12 、2.7x10 12 、2.8x10 12 、2.9x10 12 、3x10 12 、3.1x10 12 、3.2x10 12 、3.3x10 12 、3.4x10 12 、3.5x10 12 、3.6x10 12 、3.7x10 12 、3.8x10 12, 3.9x10 12 , 4x10 12 , 4.1x10 12 , 4.2x10 12 , 4.3x10 12 , 4.4x10 12 , 4.5x10 12 , 4.6x10 12 , 4.7x10 12 , 4.8x10 12 ,4.9x10 12 , 5x10 12 , 6x10 12 , 7x10 12 , 8x10 12 , 9x10 12 , 1x10 13 , 2x10 13 , 3x10 13 , 4x10 13 , 5x10 13 , 6x10 13 , 6.7x10 13 , 7x10 13 , 8x10 13 , 9x10 13 , 1x10 14 , 2x10 14 , 3x10 14 , 4x10 14 , 5x10 14 , 6x10 14 , 7x10 14 , 8x10 14 , 9x10 14 , 1x10 15 , 2x10 15 , 3x10 15 , 4x10 15 , 5x10 15 , 6x10 15 , 7x10 15 , 8x10 15 , 9x10 15 or 1x10 16 The composition concentration is VG / mL.

[0191] joint

[0192] The viral vectors comprising regulatory polynucleotides can be used in combination with one or more other therapeutic agents, prophylactic agents, diagnostic agents or imaging agents. "In combination with ... " is not intended to imply that the medicament must be used simultaneously and / or formulated for delivery together, although these delivery methods are within the scope of the present disclosure. Compositions can be used in parallel with one or more other desired therapeutic agents or medical operations, before or after them. Generally speaking, each reagent will be used with a dosage and / or time plan determined for the reagent. In certain embodiments, the present disclosure includes the delivery of a pharmaceutical composition, a prophylactic composition, a diagnostic composition or an imaging composition combined with a medicament, which can improve their bioavailability, reduce and / or modify their metabolism, inhibit their excretion, and / or modify their distribution in vivo.

[0193] deliver

[0194] In one embodiment, a viral vector comprising a regulatory polynucleotide may be administered or delivered by using the methods for delivering AAV viral particles described in European Patent Application No. EP1857552, the contents of which are incorporated herein by reference in their entirety.

[0195] In one embodiment, a viral vector comprising a regulatory polynucleotide may be administered or delivered by using the methods for delivering proteins using AAV vectors as described in European Patent Application No. EP2678433, the contents of which are incorporated herein by reference in their entirety.

[0196] In one embodiment, a viral vector comprising a regulatory polynucleotide may be administered or delivered by using the methods for delivering DNA molecules using AAV vectors as described in US Pat. No. 5,858,351, the contents of which are incorporated herein by reference in their entirety.

[0197] In one embodiment, a viral vector comprising a regulatory polynucleotide may be administered or delivered by using the methods for delivering DNA to the bloodstream described in US Pat. No. 6,211,163, the contents of which are incorporated herein by reference in their entirety.

[0198] In one embodiment, a viral vector comprising a regulatory polynucleotide may be administered or delivered by using the methods for delivering AAV viral particles described in US Pat. No. 6,325,998, the contents of which are incorporated herein by reference in their entirety.

[0199] In one embodiment, a viral vector comprising a regulatory polynucleotide may be administered or delivered by using the methods for delivering DNA to muscle cells described in US Pat. No. 6,335,011, the contents of which are incorporated herein by reference in their entirety.

[0200] In one embodiment, a viral vector comprising a regulatory polynucleotide may be administered or delivered by using the methods for delivering DNA to muscle cells and tissues described in US Pat. No. 6,610,290, the contents of which are incorporated herein by reference in their entirety.

[0201] In one embodiment, a viral vector comprising a regulatory polynucleotide may be administered or delivered by using the methods for delivering DNA to muscle cells described in US Pat. No. 7704492, the contents of which are incorporated herein by reference in their entirety.

[0202] In one embodiment, a viral vector comprising a regulatory polynucleotide may be administered or delivered by using the methods for delivering a payload to skeletal muscle described in US Pat. No. 7,112,321, the contents of which are incorporated herein by reference in their entirety.

[0203] In one embodiment, the viral vector may be administered or delivered by using the methods for delivering payloads to the central nervous system described in US Pat. No. 7,588,757, the contents of which are incorporated herein by reference in their entirety.

[0204] In one embodiment, a viral vector comprising a regulatory polynucleotide may be administered or delivered by using the methods for delivering a payload described in US Pat. No. 8,283,151, the contents of which are incorporated herein by reference in their entirety.

[0205] In one embodiment, a viral vector comprising a regulatory polynucleotide may be administered or delivered by using the methods for delivering a payload described in US Pat. No. 8,318,687, the contents of which are incorporated herein by reference in their entirety, for the treatment of Alzheimer's disease.

[0206] In one embodiment, a viral vector comprising a regulatory polynucleotide may be administered or delivered by using the methods for delivering a payload described in International Patent Publication No. WO2012144446, the contents of which are herein incorporated by reference in their entirety.

[0207] In one embodiment, a viral vector comprising a regulatory polynucleotide may be administered or delivered by using the methods for delivering a payload using a glutamate decarboxylase (GAD) delivery vector as described in International Patent Publication No. WO2001089583, the contents of which are incorporated herein by reference in their entirety.

[0208] In one embodiment, a viral vector comprising a regulatory polynucleotide may be administered or delivered by using the methods for delivering a payload described in International Patent Publication No. WO2001096587, the contents of which are incorporated herein by reference in their entirety.

[0209] In one embodiment, a viral vector comprising a regulatory polynucleotide may be administered or delivered by using the methods for delivering a payload to muscle tissue described in International Patent Publication No. WO2002014487, the contents of which are incorporated herein by reference in their entirety.

[0210] In one embodiment, a viral vector comprising a regulatory polynucleotide may be administered or delivered by using the methods for delivering a payload to neural cells described in International Patent Publication No. WO2012057363, the contents of which are herein incorporated by reference in their entirety.

[0211] The pharmaceutical compositions of the viral vectors described herein can be characterized by one or more of bioavailability, therapeutic window, and / or volume of distribution.

[0212] In one embodiment, a viral vector comprising a regulatory polynucleotide may be formulated.As a non-limiting example, the alkalinity and / or osmolality of a formulation may be optimized to ensure optimal drug distribution in the central nervous system or a region or component of the central nervous system.

[0213] In one embodiment, a viral vector comprising a regulatory polynucleotide may be delivered to a subject by a single route of administration.

[0214] In one embodiment, the viral vector comprising the regulatory polynucleotide can be delivered to the subject via a multi-site administration route. The viral vector comprising the regulatory polynucleotide can be administered to the subject at 2, 3, 4, 5 or more than 5 sites.

[0215] In one embodiment, a viral vector comprising a regulatory polynucleotide described herein may be administered to a subject using a bolus infusion.

[0216] In one embodiment, a viral vector comprising a regulatory polynucleotide described herein can be administered to a subject using sustained delivery over a period of minutes, hours, or days. The infusion rate can be varied depending on the subject, distribution, formulation, or another delivery parameter.

[0217] In one embodiment, for multi-site delivery, the catheter can be located at more than one site in the spine. The viral vector comprising the regulatory polynucleotide can be delivered with continuous and / or bolus infusion. Each delivery site can be a different quantitative administration scheme, or the same quantitative administration scheme can be used for each delivery site. As a non-limiting example, the delivery site can be in the neck and waist region. As another non-limiting example, the delivery site can be in the neck region. As another non-limiting example, the delivery site can be in the waist region.

[0218] In one embodiment, prior to delivery of a viral vector comprising a regulatory polynucleotide described herein, the subject's spinal anatomy and pathology may be analyzed. As a non-limiting example, a subject with scoliosis may have a different dosing regimen and / or catheter position than a subject without scoliosis.

[0219] In one embodiment, during delivery of the viral vector comprising the regulatory polynucleotide, the subject's spine may be oriented perpendicular to the ground.

[0220] In another embodiment, during delivery of the viral vector comprising the regulatory polynucleotide, the subject's spine may be oriented parallel to the ground.

[0221] In one embodiment, during delivery of a viral vector comprising a regulatory polynucleotide to a subject, the subject's spine may be angled relative to the ground. The angle of the subject's spine relative to the ground may be at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, or 180 degrees.

[0222] In one embodiment, the delivery method and duration are selected to provide extensive transduction in the spinal cord. As a non-limiting example, intrathecal delivery is used to provide extensive transduction along the rostral-caudal length of the spinal cord. As another non-limiting example, multi-site infusions provide a more uniform transduction along the rostral-caudal length of the spinal cord. As another non-limiting example, extended infusions provide a more uniform transduction along the rostral-caudal length of the spinal cord.

[0223] Bioavailability

[0224] When formulated into compositions with delivery / formulation agents or vehicles as described herein, viral vectors comprising the regulatory polynucleotides of the invention can exhibit increased bioavailability compared to compositions lacking delivery agents as described herein. The term "bioavailability" as used herein refers to the systemic availability of a given amount of a particular agent administered to a subject. Bioavailability is determined by measuring the area under the curve (AUC) or maximum serum or plasma concentration (C max), bioavailability can be assessed. AUC is a determination of the area under the curve that depicts the serum or plasma concentration of a compound along the ordinate (Y-axis) relative to the time along the abscissa (X-axis). Typically, the AUC of a particular compound can be calculated using methods known to those of ordinary skill in the art, and as described in GS Banker, Modern Pharmaceutics, Drugs and the Pharmaceutical Sciences, v.72, Marcel Dekker, New York, Inc., 1996 (the contents of which are incorporated herein by reference in their entirety).

[0225] C max The C value is the maximum concentration of the compound achieved in the serum or plasma of the subject after the compound is administered to the subject. The C value of a particular compound can be measured using methods known to those of ordinary skill in the art. max As used herein, the phrase "increase bioavailability" or "improve pharmacokinetics" means that the systemic availability of the viral vector of the invention can be increased in a subject (e.g., as measured by AUC, C max or C min In some embodiments, such an action may include co-administration with one or more delivery agents as described herein. In some embodiments, the bioavailability of the viral vector may be increased by at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%.

[0226] Treatment Window

[0227] Compared to the therapeutic window of the viral vector administered without one or more delivery agents as described herein, when formulated with one or more delivery agents as described herein, the viral vector comprising the regulatory polynucleotide of the present invention can show an increase in the therapeutic window of compound and / or composition administration. The term "therapeutic window" used herein means the range of plasma concentrations, or the range of levels of therapeutically active substances at the site of action, which has a high probability of causing a therapeutic effect. In certain embodiments, when administered in a formulation, the therapeutic window of the viral vector can be increased by at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or about 100%.

[0228] Distribution volume

[0229] When formulated with one or more delivery agents as described herein, viral vectors comprising a regulatory polynucleotide of the invention may exhibit improved volume of distribution (V) compared to formulations lacking one or more delivery agents as described herein. dist ), e.g., reduced or targeted. dist Relates the amount of a pharmaceutical agent in the body to the concentration of the same agent in the blood or plasma. As used herein, the term "distribution volume" means the volume of fluid required to contain the total amount of a pharmaceutical agent in the body at the same concentration as in the blood or plasma: V dist =Volume of distribution is equal to the amount of drug in the body / concentration of the drug in the blood or plasma. For example, for a 10 mg dose of a given drug and a plasma concentration of 10 mg / L, the volume of distribution would be 1 liter. The volume of distribution reflects the extent to which the drug is present in extravascular tissues. A large volume of distribution reflects the tendency of the drug to bind to tissue components relative to plasma proteins. In clinical settings, V dist Can be used to determine the loading dose to achieve steady-state concentration. In certain embodiments, when co-administered with one or more delivery agents as described herein, the volume of distribution of the viral vector composition of the invention can be reduced by at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%.

[0230] Kits and Devices

[0231] The present invention provides a variety of kits for conveniently and / or effectively implementing the methods of the present invention. Typically, the kit will contain sufficient amounts and / or numbers of components to allow the user to perform multiple treatments of a subject and / or perform multiple experiments.

[0232] Any of the vectors, constructs, regulatory polynucleotides, polynucleotides or polypeptides of the present invention may be included in a kit. In certain embodiments, the kit may also include reagents and / or instructions for creating and / or synthesizing the compounds and / or compositions of the present invention. In certain embodiments, the kit may also include one or more buffers. In certain embodiments, the kit of the present invention may include components for preparing protein or nucleic acid arrays or libraries, and thus, may include, for example, solid supports.

[0233] In certain embodiments, the kit components can be packaged in aqueous media or in lyophilized form. The container device of the kit generally includes at least one vial, test tube, flask, bottle, syringe or other container device, in which the components can be placed, and preferably, appropriately divided. In the case of more than one kit component (labeling reagents and labels can be packaged together), the kit can also generally contain a second container, a third container or other additional container, in which the additional components can be placed separately. In certain embodiments, the kit can also include a second container device for accommodating sterile, pharmaceutically acceptable buffer and / or other diluents. In certain embodiments, different combinations of components can be included in one or more vials. The kit of the present invention can also generally include a device for accommodating the compound and / or composition of the present invention, for example, strictly restricting commercially sold proteins, nucleic acids and any other reagent containers. Such a container can include an injection or blow-molded plastic container, in which the desired vial is retained.

[0234] In certain embodiments, kit components are provided in one and / or multiple liquid solutions. In certain embodiments, the liquid solution is an aqueous solution, wherein a sterile aqueous solution is particularly preferred. In certain embodiments, kit components can be provided as dry powders. When reagents and / or components are provided as dry powders, such powders can be reconstructed by adding a solvent of suitable volume. In certain embodiments, it is foreseen that a solvent can also be provided in another container device. In certain embodiments, a marker dye is provided as a dry powder. In certain embodiments, it is foreseen that 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000 micrograms or at least or at most the dry dye of those amounts are provided in the kit of the present invention. In such embodiments, the dye may then be resuspended in any suitable solvent, such as DMSO.

[0235] In certain embodiments, the kit may include instructions for use of the kit components as well as any additional reagents not included in the kit. The instructions may include possible changes that may be implemented.

[0236] Device

[0237] In certain embodiments, the compounds and / or compositions of the invention can be combined with a device, coated on a device, or embedded in a device. The device can include, but is not limited to, a dental implant, a stent, a bone replacement, an artificial joint, a valve, a pacemaker, and / or other implantable therapeutic devices.

[0238] The present invention provides devices that can contain viral vectors encoding one or more regulatory polynucleotide payload molecules. These devices contain viral vectors in a stable formulation that can be immediately delivered to a subject in need thereof, such as a human patient.

[0239] Devices for administration may be employed to deliver viral vectors comprising a regulatory polynucleotide of the invention according to single, multiple, or divided dosing schedules taught herein.

[0240] It is contemplated that methods and devices known in the art for multiple administrations to cells, organs, and tissues are used in conjunction with the methods and compositions disclosed herein as embodiments of the invention. These include, for example, those methods and devices with multiple needles, hybrid devices employing, for example, lumens or catheters, and devices utilizing heat, current, or radiation actuation mechanisms.

[0241] The regulatory polynucleotides of the invention may be used to treat, prevent or ameliorate any disease or disorder characterized by aberrant or undesirable target expression.

[0242] definition

[0243] At various places in the present specification, substituents of compounds of the present disclosure are disclosed in groups or in ranges. The present disclosure is specifically intended to include each and every individual subcombination of the members of such groups and ranges.

[0244] About: As used herein, the term "about" refers to ± 10% of the stated value.

[0245] Co-administration: As used herein, the term "co-administration" or "combined administration" refers to the administration of two or more agents to a subject simultaneously or over a period of time such that there may be overlap in the effects of each agent on the patient. In certain embodiments, they are administered within about 60, 30, 15, 10, 5, or 1 minute of each other. In certain embodiments, the administration of the agents is scheduled closely enough to achieve a combined (e.g., synergistic) effect.

[0246] Animal: The term "animal" as used herein refers to any member of the animal kingdom. In certain embodiments, "animal" refers to a person at any stage of development. In certain embodiments, "animal" refers to a non-human animal at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, a cow, a primate, or a pig). In certain embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and worms. In certain embodiments, the animal is a transgenic animal, a genetically engineered animal, or a clone.

[0247] Approximately: As used herein, the term "approximately" or "about" applied to one or more target values ​​represents a value similar to the reference value. In certain embodiments, the term "approximately" or "about" represents a range of values ​​falling within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (greater than or less than) of the reference value, unless otherwise stated or otherwise apparent from the context (except where such a number would exceed 100% of the possible value).

[0248] As used herein, the terms "associated with," "conjugated," "connected," "attached," and "tethered," when used with respect to two or more moieties, mean that the moieties are physically bound or linked to one another (either directly or through one or more additional moieties that act as linkers) to form a structure that is sufficiently stable such that the moieties remain physically bound under the conditions (e.g., physiological conditions) under which the structure is used. "Binding" need not be strictly by direct covalent chemical bonding. It may also imply a connection based on ionic or hydrogen bonding or hybridization that is sufficiently stable such that the "bound" entities remain physically bound.

[0249] Bifunctional: As used herein, the term "bifunctional" refers to any substance, molecule, or moiety that is capable of performing or maintaining at least two functions. The functions may affect the same result or different results. The structures that produce the functions may be the same or different.

[0250] Biocompatible: As used herein, the term "biocompatible" means compatible with living cells, tissues, organs, or systems, producing little to no risk of injury, toxicity, or rejection by the immune system.

[0251] Biodegradable: As used herein, the term "biodegradable" means capable of being broken down into harmless products by the action of living organisms.

[0252] Biologically active: As used herein, the phrase "biologically active" refers to the characteristic of any substance that is active in a biological system and / or organism. For example, a substance that has a biological effect on an organism when administered to the organism is considered biologically active. In certain embodiments, a regulatory polynucleotide of the invention may be considered biologically active if even a portion of the polynucleotide is biologically active or mimics an activity that is considered biologically relevant.

[0253] Induced pluripotent stem cell: As used herein, an "induced pluripotent stem cell" is a cell that can be induced to form any of several different cell types.

[0254] Compound: As used herein, the term "compound" is intended to include all stereoisomers, geometric isomers, tautomers, and isotopes of the depicted structure.

[0255] The compounds described herein may be asymmetric (e.g., have one or more stereocenters). All stereoisomers, such as enantiomers and diastereomers, are contemplated unless otherwise indicated. Compounds of the disclosure containing asymmetrically substituted carbon atoms may be isolated in optically active or racemic form. Methods of preparing optically active forms from optically active starting materials are known in the art, such as by resolving racemic mixtures or by stereoselective synthesis. Many geometric isomers of alkenes, C=N double bonds, etc. may also be present in the compounds described herein, and all such stable isomers are contemplated in the present disclosure. Cis and trans geometric isomers of the compounds of the present disclosure are described, and they may be isolated as mixtures of isomers or as separated isomeric forms.

[0256] The compounds of the present disclosure also include tautomeric forms. Tautomeric forms result from the swapping of a single bond with an adjacent double bond and the concomitant migration of a proton. Tautomeric forms include prototropic tautomers, which are isomeric protonation states having the same empirical formula and total charge.

[0257] Compounds of the present disclosure also include all isotopes of atoms that occur in intermediate compounds or final compounds. "Isotopes" refer to atoms that have the same atomic number but have different mass numbers caused by different neutron numbers in the nucleus. For example, isotopes of hydrogen include tritium and deuterium.

[0258] The compounds and salts of the present disclosure can be prepared by combining with solvents or water molecules to form solvates and hydrates by conventional methods.

[0259] Conserved: The term "conserved" as used herein refers to nucleotides or amino acid residues of each polynucleotide sequence or polypeptide sequence that are present without change in the same position of two or more sequences being compared. Relatively conserved nucleotides or amino acids are those that are conserved in more related sequences compared to nucleotides or amino acids that occur elsewhere in the sequence.

[0260] In certain embodiments, if two or more sequences are 100% identical to each other, they are said to be "fully conserved". In certain embodiments, if two or more sequences are at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical to each other, they are said to be "highly conserved". In certain embodiments, if two or more sequences are about 70% identical, about 80% identical, about 90% identical, about 95%, about 98%, or about 99% identical to each other, they are said to be "highly conserved". In certain embodiments, if two or more sequences are at least 30% identical, at least 40% identical, at least 50% identical, at least 60% identical, at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical to each other, they are said to be "conserved". In certain embodiments, two or more sequences are said to be "conserved" if they are about 30% identical, about 40% identical, about 50% identical, about 60% identical, about 70% identical, about 80% identical, about 90% identical, about 95% identical, about 98% identical, or about 99% identical to each other. Conservation of sequence can apply to the entire length of a polynucleotide or polypeptide, or can apply to portions, regions, or components thereof.

[0261] Controlled release: As used herein, the term "controlled release" refers to the release characteristics of a pharmaceutical composition or compound that conforms to a specific release profile to achieve a therapeutic result.

[0262] Cyclic or cyclized: As used herein, the term "cyclic" refers to the presence of continuous rings. Circular molecules need not be circular, merely linked to form a continuous chain of subunits.

[0263] Cytostatic: As used herein, "cytostatic" means inhibiting, reducing, suppressing the growth, division, or reproduction of cells (e.g., mammalian cells (e.g., human cells)), bacteria, viruses, fungi, protozoa, parasites, prions, or combinations thereof.

[0264] Cytotoxic: As used herein, "cytotoxic" means killing or causing an injurious, poisonous, or lethal effect to cells (eg, mammalian cells (eg, human cells)), bacteria, viruses, fungi, protozoa, parasites, prions, or combinations thereof.

[0265] Delivery: As used herein, "delivery" means the act or manner of delivering a compound, substance, entity, moiety, load or payload.

[0266] Delivery agent: As used herein, "delivery agent" refers to any substance that at least partially facilitates the delivery of a regulatory polynucleotide to a targeted cell in vivo.

[0267] Destabilized: As used herein, the terms "destabilizable," "destabilizing," or "destabilizing region" refer to a region or molecule that is less stable than the starting, wild-type or native form of the same region or molecule.

[0268] Detectable marker: As used herein, "detectable marker" refers to one or more markers, signals or moieties that are attached, incorporated or bound to another entity and that are readily detected by methods known in the art, including radiography, fluorescence, chemiluminescence, enzyme activity, absorbance, and the like. Detectable markers include radioisotopes, fluorophores, chromophores, enzymes, dyes, metal ions, ligands such as biotin, avidin, streptavidin and haptens, quantum dots, and the like. Detectable markers may be located at any position of the peptides or proteins disclosed herein. They may be within an amino acid, peptide or protein, or located at the N-terminus or C-terminus.

[0269] Diastereoisomers: As used herein, the term "diastereoisomers" refers to stereoisomers that are not mirror images of each other and are not superimposable on each other.

[0270] Digestion: As used herein, the term "digestion" means breaking down into smaller pieces or components. When referring to polypeptides or proteins, digestion results in the production of peptides.

[0271] Distal: As used herein, the term "distal" refers to located away from the center or away from a target point or area.

[0272] Dosing regimen: As used herein, a "dosing regimen" is a regimen of administration planned or determined by a physician for therapeutic, preventive, or palliative therapy.

[0273] Enantiomer: As used herein, the term "enantiomer" refers to each individual optically active form of a compound of the invention having an optical purity or enantiomeric excess (determined by standard methods in the art) of at least 80% (i.e., at least 90% of one enantiomer and up to 10% of the other enantiomer), preferably at least 90% and more preferably at least 98%.

[0274] Encapsulate: As used herein, the term "encapsulate" means to surround, enclose, or wrap.

[0275] Engineered: As used herein, an embodiment of the invention is "engineered" when the embodiment is designed to have structural or chemical characteristics or properties that differ from the starting, wild-type or native molecule.

[0276] Effective amount: As used herein, an "effective amount" of an agent is an amount sufficient to achieve a beneficial or desired result, e.g., a clinical result, and as such, an "effective amount" depends on the context in which it is used. For example, in the context of administering an agent to treat cancer, an effective amount of an agent is, e.g., an amount sufficient to achieve treatment of the cancer as defined herein, as compared to the response obtained without administration of the agent.

[0277] Exosome: As used herein, an "exosome" is a vesicle or complex secreted by mammalian cells that is involved in RNA degradation.

[0278] Expression: As used herein, "expression" of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of the RNA transcript (e.g., by splicing, editing, 5' cap formation and / or 3' end processing); (3) translation of the RNA into a polypeptide or protein; and (4) post-translational modification of the polypeptide or protein.

[0279] Characteristic: As used herein, "characteristic" means a characteristic, property, or distinctive element.

[0280] Formulation: As used herein, "formulation" comprises at least one regulatory polynucleotide and a delivery agent.

[0281] Fragment: As used herein, "fragment" means a portion. For example, a fragment of a protein may include a polypeptide obtained by digestion of a full-length protein isolated from cultured cells.

[0282] Functional: As used herein, a "functional" biomolecule is a biomolecule in a form in which it exhibits the properties and / or activities that characterize it.

[0283] Homology: The term "homology" as used herein refers to the overall relatedness between polymer molecules, such as nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In certain embodiments, if the sequences of polymer molecules are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical or similar, they are considered to be "homologous" to each other. The term "homologous" necessarily refers to the comparison between at least two sequences (polynucleotide or polypeptide sequences). According to the present invention, if the polypeptides encoded by two polynucleotide sequences are at least about 50%, 60%, 70%, 80%, 90%, 95% or even 99% identical for at least one fragment of at least about 20 amino acids, the two polynucleotide sequences are considered to be homologous. In certain embodiments, homologous polynucleotide sequences are characterized by the ability to encode fragments of at least 4-5 uniquely specified amino acids. For polynucleotide sequences less than 60 nucleotides in length, homology is determined by the ability to encode fragments of at least 4-5 uniquely specified amino acids. According to the present invention, two protein sequences are considered homologous if they are at least about 50%, 60%, 70%, 80% or 90% identical for at least one fragment of at least about 20 amino acids.

[0284] Identity: The term "identity" as used herein refers to the overall relatedness between polymer molecules, such as between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. For example, the calculation of the percent identity of two polynucleotide sequences can be performed as follows: the two sequences are aligned for the purpose of optimal alignment (e.g., gaps can be introduced in one or both of the first and second nucleic acid sequences for optimal alignment, and non-identical sequences can be discarded for comparison purposes). In certain embodiments, the length of the sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide at the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences. Mathematical algorithms can be used to achieve sequence comparison and identity percentage determination between two sequences. For example, the identity percentage between two nucleotide sequences can be determined using a variety of methods, such as those described in the following documents: Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, ed., Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., ed., M Stockton Press, New York, 1991; each of which is incorporated herein by reference.For example, using the algorithm of Meyers and Miller (CABIOS, 1989, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weighted residue table, a gap length penalty of 12, and a gap penalty of 4, the percent identity between two nucleotide sequences can be determined. Alternatively, using the GAP program in the GCG software package, using the NWSgapdna.CMP matrix, the percent identity between two nucleotide sequences can be determined. Methods commonly used to determine the percent identity between sequences include, but are not limited to, those disclosed in Carillo, H., and Lipman, D., SIAMJ Applied Math., 48: 1073 (1988); the document is incorporated herein by reference. In addition, techniques for determining identity are codified in publicly available computer programs. Exemplary computer software for determining homology between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research, 12(1), 387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, SF et al., J. Molec. Biol., 215, 403 (1990)).

[0285] Inhibit the expression of a gene: As used herein, the phrase "inhibit the expression of a gene" refers to causing a reduction in the amount of an expression product of a gene. The expression product may be an RNA (e.g., mRNA) transcribed from a gene or a polypeptide translated from an RNA transcribed from a gene. Typically, a reduction in the level of mRNA results in a reduction in the level of a polypeptide translated therefrom. The level of expression may be determined using standard techniques for measuring mRNA or protein.

[0286] Isomer: The term "isomer" as used herein refers to any tautomer, stereoisomer, enantiomer or diastereomer of any compound of the present invention. It should be recognized that the compounds of the present invention may have one or more chiral centers and / or double bonds, and therefore, exist as stereoisomers, such as double bond isomers (i.e., geometric E / Z isomers) or diastereomers (e.g., enantiomers (i.e., (+) or (-)) or cis / trans isomers). According to the present invention, the chemical structures described herein, and therefore the compounds of the present invention, include all corresponding stereoisomers, that is, stereoisomerically pure forms (e.g., geometrically pure, enantiomerically pure or diastereomerically pure) and enantiomeric and stereoisomeric mixtures, e.g., racemates. Enantiomeric and stereoisomeric mixtures of the compounds of the invention can generally be resolved into their component enantiomers or stereoisomers by well-known methods, such as chiral gas chromatography, chiral high performance liquid chromatography, crystallization of the compounds as chiral salt complexes or crystallization of the compounds in chiral solvents. Enantiomers and stereoisomers can also be obtained from stereoisomerically or enantiomerically pure intermediates, reagents and catalysts by well-known asymmetric synthetic methods.

[0287] In vitro: As used herein, the term "in vitro" refers to events that occur in an artificial environment, such as in a test tube or reaction vessel, in a cell culture, in a petri dish, etc., rather than within an organism (e.g., an animal, plant, or microorganism).

[0288] In vivo: As used herein, the term "in vivo" refers to events that occur within an organism (eg, an animal, plant, or microorganism, or a cell or tissue thereof).

[0289] Separated: The term "separated" as used herein means a substance or entity that has been separated from at least some of the components that accompany it (whether in nature or in an experimental setting). With reference to the substances that have been combined with them, the separated substances can have different purity levels. The separated substances and / or entities can be separated from other components that were originally combined with them by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or more. In certain embodiments, the separated medicament has a purity exceeding about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or exceeding about 99%. As used herein, if a substance does not contain other components substantially, it is "pure".

[0290] Substantially isolated: "Substantially isolated" means that the compound is substantially separated from the environment in which it is formed or detected. Partial separation can include, for example, a composition enriched with a compound of the disclosure. Substantially isolated can include a composition containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% by weight of a compound of the disclosure or a salt thereof. Methods for separating compounds and their salts are conventional in the art.

[0291] Linker: As used herein, a linker refers to a group of atoms, for example, 10-1,000 atoms, and may include atoms or groups, for example, but not limited to, carbon, amino, alkylamino, oxygen, sulfur, sulfoxide, sulfonyl, carbonyl, and imine. The linker may be connected to a modified nucleoside or nucleotide on a nucleobase or sugar moiety at a first end, and to a payload, for example, a detectable or therapeutic agent, at a second end. The linker may have a sufficient length so as not to interfere with incorporation into a nucleic acid sequence. The linker may be used for any useful purpose, such as forming a regulatory polynucleotide polymer (e.g., by connecting two or more regulatory polynucleotide molecules) or a regulatory polynucleotide conjugate as described herein, and administering a payload. Examples of chemical groups that may be inserted into a linker include, but are not limited to, alkyl, alkenyl, alkynyl, amido, amino, ether, thioether, ester, alkylene, heteroalkylene, aryl, or heterocyclic groups, each of which may be optionally substituted as described herein. Examples of linkers include, but are not limited to, unsaturated alkanes, polyethylene glycols (e.g., ethylene glycol or propylene glycol monomer units, e.g., diethylene glycol, dipropylene glycol, triethylene glycol, tripropylene glycol, tetraethylene glycol or tetraethylene glycol), and dextran polymers and derivatives thereof. Other examples include, but are not limited to, cleavable moieties within the linker, e.g., disulfide bonds (-SS-) or azo bonds (-N=N-), which can be cleaved using reducing agents or photolysis. Non-limiting examples of selectively cleavable bonds include amide bonds that can be cleaved, e.g., by using tris(2-carboxyethyl)phosphine (TCEP) or other reducing agents and / or photolysis, and ester bonds that can be cleaved, e.g., by acidic or basic hydrolysis.

[0292] MicroRNA (miRNA) binding site: As used herein, a microRNA (miRNA) binding site refers to a nucleotide position or region of a nucleic acid transcript to which at least the "seed" region of a miRNA binds.

[0293] Modified: As used herein, "modified" refers to an altered state or structure of a molecule of the invention. Molecules can be modified in many ways, including chemically, structurally, and functionally.

[0294] Naturally occurring: As used herein, "naturally occurring" means occurring in nature without human assistance.

[0295] Neutralizing antibody: As used herein, "neutralizing antibody" refers to an antibody that binds to its antigen and defends cells from the antigen or infectious agent (by neutralizing or eliminating any biological activity it has).

[0296] Non-human vertebrates: As used herein, "non-human vertebrates" include all vertebrates other than Homo sapiens, including wild and domesticated species. Examples of non-human vertebrates include, but are not limited to, mammals such as alpacas, gaur, bison, camels, cats, cattle, deer, dogs, donkeys, steers, goats, guinea pigs, horses, llamas, mules, pigs, rabbits, reindeer, sheep, buffalo, and yaks.

[0297] Off-target: As used herein, "off-target" refers to any unintended effect on any one or more targets, genes, or cellular transcripts.

[0298] Open reading frame: As used herein, "open reading frame" or "ORF" refers to a sequence that does not contain a stop codon in a given reading frame.

[0299] Operably linked: As used herein, the phrase "operably linked" means a functional linkage between two or more molecules, constructs, transcripts, entities, moieties, etc.

[0300] Optionally substituted: A phrase of the form "optionally substituted X" (e.g., optionally substituted alkyl) is intended herein to be equivalent to "X, wherein X is optionally substituted" (e.g., "alkyl, wherein the alkyl is optionally substituted"). It is not intended to indicate that the feature "X" (e.g., alkyl) itself is optional.

[0301] Peptide: As used herein, a "peptide" has a length of less than or equal to 50 amino acids, eg, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

[0302] Patient: As used herein, "patient" means a subject who may seek or need treatment, requires treatment, is receiving treatment, will receive treatment, or is under the care of a trained professional for a particular disease or condition.

[0303] Pharmaceutically acceptable: The phrase "pharmaceutically acceptable" is used herein to refer to compounds, substances, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0304] Pharmaceutically acceptable excipients: As used herein, the phrase "pharmaceutically acceptable excipients" means any ingredient other than the compounds described herein (e.g., a vehicle capable of suspending or dissolving the active compound) and which has substantially non-toxic and non-inflammatory properties in patients. Excipients may include, for example, anti-adherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colorants), softeners, emulsifiers, fillers (diluents), film formers or coatings, flavoring agents, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and water of hydration. Exemplary excipients include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dicalcium phosphate), calcium stearate, croscarmellose, cross-linked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[0305] Pharmaceutically acceptable salts: The present disclosure also includes pharmaceutically acceptable salts of the compounds described herein. As used herein, "pharmaceutically acceptable salts" refer to derivatives of the disclosed compounds in which the parent compound is modified by converting an existing acid or base moiety into its salt form (e.g., by reacting a free base group with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. Representative acid addition salts include acetate, acetic acid, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzenesulfonic acid, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate, and the like. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc., and non-toxic ammonium, quaternary ammonium and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, etc. The pharmaceutically acceptable salts of the present disclosure include conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present disclosure can be synthesized from the parent compound containing a basic or acidic part by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of an appropriate base or acid in water or an organic solvent or a mixture of the two; generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred.Lists of suitable salts can be found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418, Pharmaceutical Salts: Properties, Selection, and Use, PH Stahl and CG Wermuth (Eds.), Wiley-VCH, 2008, and Berge et al., Journal of Pharmaceutical Science, 66, 1-19 (1977), each of which is incorporated herein by reference in its entirety.

[0306] Pharmaceutically acceptable solvate: The term "pharmaceutically acceptable solvate" as used herein refers to a compound of the present invention in which molecules of a suitable solvent are incorporated into the crystal lattice. Suitable solvents are physiologically tolerable at the doses administered. For example, solvates can be prepared by crystallization, recrystallization or precipitation from a solution comprising an organic solvent, water or a mixture thereof. Examples of suitable solvents are ethanol, water (e.g., monohydrate, dihydrate and trihydrate), N-methylpyrrolidone (NMP), dimethyl sulfoxide (DMSO), N,N'-dimethylformamide (DMF), N,N'-dimethylacetamide (DMAC), 1,3-dimethyl-2-imidazolidinone (DMEU), 1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone (DMPU), acetonitrile (ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone, benzyl benzoate, etc. When water is the solvent, the solvate is referred to as a "hydrate".

[0307] Pharmacokinetics: As used herein, "pharmacokinetics" refers to any one or more properties of a molecule or compound as it relates to determining the fate of a substance administered to a living organism. Pharmacokinetics is divided into several areas, including the extent and rate of absorption, distribution, metabolism, and excretion. This is generally referred to as ADME, where: (A) absorption is the process by which a substance enters the blood circulation; (D) distribution is the dispersion or spread of a substance throughout the body's fluids and tissues; (M) metabolism (or biotransformation) is the irreversible conversion of a parent compound into daughter metabolites; and (E) excretion (or elimination) refers to the elimination of a substance from the body. In rare cases, some drugs accumulate irreversibly in body tissues.

[0308] Physicochemical: As used herein, "physicochemical" means or relates to physical and / or chemical properties.

[0309] Prevention: As used herein, the term "prevention" means partially or completely delaying the onset of an infection, disease, disorder, and / or condition; partially or completely delaying the onset of one or more symptoms, features, or clinical manifestations of a specific infection, disease, disorder, and / or condition; partially or completely delaying the onset of one or more symptoms, features, or manifestations of a specific infection, disease, disorder, and / or condition; partially or completely delaying the progression from an infection, a specific disease, disorder, and / or condition; and / or reducing the risk of developing conditions associated with an infection, disease, disorder, and / or condition.

[0310] Prodrugs: The present disclosure also includes prodrugs of the compounds described herein. As used herein, "prodrug" means any substance, molecule, or entity in a form that is predicted to act as a therapeutic agent after chemical or physical changes. The prodrug may be covalently bound or masked in some manner and released or converted to an active drug moiety before, during, or after administration to a mammalian subject. Prodrugs may be prepared by modifying functional groups present in a compound in a particular manner such that the modification is cleaved into the parent compound during routine manipulation or in vivo. Prodrugs include compounds in which a hydroxyl, amino, sulfhydryl, or carboxyl group is bonded to any group that, when administered to a mammalian subject, cleaves to form a free hydroxyl, amino, sulfhydryl, or carboxyl group, respectively. The preparation and use of prodrugs are discussed in T. Higuchi and V. Stella, "Pro-drugs as Novel Delivery Systems," Volume 14 of the ACSSymposium Series and Bioreversible Carriers in Drug Design, Edward B. Roche, ed., American Pharmaceutical Association and Pergamon Press, 1987, both of which are hereby incorporated by reference in their entirety. In certain embodiments, the primary-miR of the present invention can be a prodrug of the pre-miR. Similarly, the primary-miR or pre-miR can be a prodrug of the artificial miR processed therefrom.

[0311] Proliferation: As used herein, the term "proliferation" means to grow, expand or increase rapidly or to cause growth, expansion or increase. "Proliferative" means having the ability to proliferate. "Antiproliferative" means having the property of resisting or counteracting proliferative properties.

[0312] Prophylactic: As used herein, "prophylactic" refers to a therapeutic agent or course of action used to prevent the spread of disease.

[0313] Prevention: As used in this article, “prevention” means measures taken to maintain health and stop the spread of disease.

[0314] Protein cleavage site: As used herein, "protein cleavage site" refers to a site at which controlled cleavage of an amino acid chain can be accomplished chemically, enzymatically, or photochemically.

[0315] Protein cleavage signal: As used herein, "protein cleavage signal" means at least one amino acid that tags or marks a polypeptide for cleavage.

[0316] Target protein: The term "target protein" or "desired protein" as used herein includes those provided herein and fragments, mutants, variants and alterations thereof.

[0317] Proximal: As used herein, the term "proximal" means located near the center or near a target point or area.

[0318] Purify: As used herein, "purify," "purified," means rendering substantially pure or free from undesirable components, contaminants, admixtures, or impurities.

[0319] Sample: As used herein, the term "sample" or "biological sample" refers to a subset of its tissues, cells or components (e.g., body fluids, including but not limited to blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid, and semen). A sample may also include a homogenate, lysate, or extract prepared from a whole organism or a subset of its tissues, cells or components, or a fraction or part thereof, including but not limited to, for example, plasma, serum, cerebrospinal fluid, lymph, external parts of the skin, respiratory tract, intestinal tract, and urogenital tract, tears, saliva, milk, blood cells, tumors, organs. A sample also refers to a culture medium, such as a nutrient broth or gel, which may contain cellular components, such as proteins or nucleic acid molecules.

[0320] Signal sequence: As used herein, the phrase "signal sequence" refers to a sequence that can direct the trafficking or localization of a protein.

[0321] Single unit dose: As used herein, a "single unit dose" is a dose of any therapeutic agent administered at one dose / at one time / single route / single contact point (ie, a single administration event).

[0322] Similarity: The term "similarity" as used herein refers to the overall relatedness between polymer molecules, such as between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. The percentage of similarity between polymer molecules can be calculated in the same manner as the calculation of percentage identity, except that, as understood in the art, the calculation of percentage similarity takes into account conservative substitutions.

[0323]

[00136] Split dose: As used herein, "split dose" is the division of a single unit dose or total daily dose into two or more doses.

[0324] Stable: As used herein, "stable" refers to a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and preferably capable of formulation into an efficacious therapeutic agent.

[0325] Stabilized: As used herein, the terms "stabilize," "stabilized," "stabilized region," mean making or becoming stable.

[0326] Stereoisomers: The term "stereoisomer" as used herein refers to all possible different isomeric and conformational forms that a compound (e.g., a compound of any formula described herein) may have, especially all possible stereochemical and conformational isomeric forms, all diastereomers, enantiomers and / or conformational isomers of the basic molecular structure. Some compounds of the present invention may exist in different tautomeric forms, which are all included within the scope of the present invention.

[0327] Subject: As used herein, the term "subject" or "patient" refers to any organism to which a composition according to the present invention can be administered, e.g., for experimental, diagnostic, preventive and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and / or plants.

[0328] Substantially: The term "substantially" as used herein refers to the qualitative condition of exhibiting a total or nearly total degree or degree of a target characteristic or property. One of ordinary skill in the biological field will understand that biological and chemical phenomena rarely, if ever, reach completion and / or advance to completeness or achieve or avoid an absolute result. The term "substantially" is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0329] Substantially equal to: As used herein, this term refers to plus / minus 2% when referring to the time difference between doses.

[0330] Substantially simultaneously: As used herein, when referring to multiple doses, this term means within 2 seconds.

[0331] Suffering from: An individual who is "suffering from" a disease, disorder, and / or condition has been diagnosed with or exhibits one or more symptoms of the disease, disorder, and / or condition.

[0332] Susceptible: An individual who is "susceptible" to a disease, disorder, and / or condition has not yet been diagnosed and / or may not exhibit symptoms of the disease, disorder, and / or condition, but contains a predisposition to develop the disease or its symptoms. In certain embodiments, an individual who is susceptible to a disease, disorder, and / or condition (e.g., cancer) may be characterized by one or more of the following: (1) a genetic mutation associated with the development of the disease, disorder, and / or condition; (2) a genetic polymorphism associated with the development of the disease, disorder, and / or condition; (3) increased and / or decreased expression and / or activity of proteins and / or nucleic acids associated with the disease, disorder, and / or condition; (4) habits and / or lifestyles associated with the development of the disease, disorder, and / or condition; (5) a family history of the disease, disorder, and / or condition; and (6) exposure to and / or infection by a microorganism associated with the development of the disease, disorder, and / or condition. In certain embodiments, an individual who is susceptible to a disease, disorder, and / or condition will develop the disease, disorder, and / or condition. In certain embodiments, an individual who is susceptible to a disease, disorder, and / or condition will not develop the disease, disorder, and / or condition.

[0333] Sustained release: As used herein, the term "sustained release" refers to a release profile of a pharmaceutical composition or compound that is consistent with a release rate over a specific period of time.

[0334] Synthetic: The term "synthetic" means produced, prepared and / or manufactured by human hand. The synthesis of polynucleotides or polypeptides or other molecules of the invention may be chemical or enzymatic.

[0335] Targeted cells: As used herein, "targeted cells" refers to any one or more target cells. The cells may be in vitro, in vivo, in situ, or in a tissue or organ of an organism. The organism may be an animal, preferably a mammal, more preferably a human, and most preferably a patient.

[0336] Therapeutic agent: The term "therapeutic agent" means any agent that has a therapeutic, diagnostic, and / or prophylactic effect and / or induces a desired biological and / or pharmacological effect when administered to a subject.

[0337] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" refers to the amount of an agent (e.g., a nucleic acid, a drug, a therapeutic agent, a diagnostic agent, a prophylactic agent, etc.) to be delivered, which, when administered to a subject suffering from or susceptible to an infection, disease, disorder, and / or condition, is sufficient to treat, diagnose, prevent, ameliorate the symptoms of, and / or delay the onset of the infection, disease, disorder, and / or condition.

[0338] Therapeutically effective outcome: As used herein, the term "therapeutically effective outcome" refers to an outcome that is sufficient to treat, diagnose, prevent, ameliorate symptoms of, and / or delay the onset of an infection, disease, disorder, and / or condition in a subject suffering from or susceptible to the infection, disease, disorder, and / or condition.

[0339] Total daily dose: As used herein, "total daily dose" is the amount given or prescribed over a 24-hour period. It may be administered as a single unit dose.

[0340] Transfection: The term "transfection" as used herein refers to a method of introducing exogenous nucleic acid into a cell. Transfection methods include, but are not limited to, chemical methods, physical treatments, and cationic lipids or mixtures.

[0341] Treat: As used herein, the term "treat" means partially or completely alleviating, ameliorating, improving, relieving, delaying the onset of, inhibiting the progression of, reducing the severity of, and / or reducing the incidence of one or more symptoms or features of a particular infection, disease, disorder, and / or condition. For example, "treating" cancer can mean inhibiting the survival, growth, and / or spread of a tumor. Treatment can be administered to a subject who does not show signs of a disease, disorder, and / or condition, and / or to a subject who only shows early signs of a disease, disorder, and / or condition, with the goal of reducing the risk of developing conditions associated with the disease, disorder, and / or condition.

[0342] Unmodified: As used herein, "unmodified" refers to any substance, compound, or molecule prior to being altered in any way. Unmodified can, but does not always, refer to the wild-type or native form of a biomolecule. A molecule can undergo a series of modifications, whereby each modified molecule can serve as an "unmodified" starting molecule for subsequent modifications.

[0343] Equivalent programs and scope

[0344] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments of the invention described herein.The scope of the present invention is not intended to be limited to the above description, but rather is set forth in the appended claims.

[0345] In the claims, articles such as "a", "an", and "the" may refer to one or more than one, unless indicated to the contrary or otherwise obvious from the context. Claims or descriptions including "or" between one or more members of a group are deemed to be satisfied if one, more than one, or all of the members of the group are present in, used in, or otherwise related to a specified product or process, unless indicated to the contrary or otherwise obvious from the context. The invention includes embodiments in which exactly one member of the group is present in, used in, or otherwise related to a given product or method. The invention includes embodiments in which more than one or all of the members of the group are present in, used in, or otherwise related to a given product or method.

[0346] It should also be noted that the term "comprising" is intended to be open ended and allows but does not require the inclusion of additional elements or steps. When the term "comprising" is used herein, the term "consisting of" is also included and disclosed.

[0347] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Methods and materials used in the present disclosure are described herein; other suitable methods and materials known in the art can also be used.

[0348] In the case of giving a range, the endpoints are included. In addition, it should be understood that, unless otherwise indicated or otherwise obvious from the context and the understanding of a person of ordinary skill in the art, the numerical values ​​expressed in the range can present any specific numerical value or sub-range within the range in different embodiments of the present invention, which is accurate to one tenth of the lower limit unit of the range, unless the context clearly indicates otherwise.

[0349] In addition, it should be understood that any specific embodiment of the present invention that falls within the prior art can be explicitly excluded from any one or more claims. Because such embodiments are considered to be known to those of ordinary skill in the art, they can be excluded, even if the exclusion is not explicitly set forth herein. Any specific embodiment of the composition of the present invention (e.g., any nucleic acid or protein encoded therefrom; any production method; any method of use, etc.) can be excluded from any one or more claims for any reason, whether or not related to the existence of the prior art.

[0350] All cited sources, e.g., references, publications, databases, database entries, and papers cited herein, are incorporated by reference into this application, even if not explicitly stated in the citation. In the event of a conflict between the description of a cited source and this application, the description in this application shall control.

[0351] The section and table headings are not intended to be limiting.

[0352] Example

[0353] Example 1. Design of regulatory polynucleotides (artificial primary- or pre-microRNA)

[0354] The artificial primary- or pre-microRNA is designed as an shRNA or stem-loop structure encoding an artificial miR (or artificial siRNA) having at least one strand that can at least partially hybridize to a target nucleic acid (e.g., RNA or DNA) and one or more of the following features: (a) a UG motif at the base of the base stem, (b) an UGUG motif at the 5' end of the miRNA loop, (c) a uridine at the 5' end of the guide strand, (d) a loop structure derived from a classical microRNA such as miR-22, (e) a CNNC at the 3' flanking sequence, (f) a flanking region from a classical microRNA such as let-7b, and / or (g) one or more bulges and mismatches such as between the passenger and guide strands.

[0355] After design, the sequence is engineered or synthesized or inserted into a plasmid or vector and administered to a cell or organism. A suitable plasmid or vector is any one that transduces or transfects a target cell.

[0356] Adeno-associated viral vectors (AAV), viral particles or whole viruses may be used.

[0357] Administration results in processing of the regulatory polynucleotide to produce an artificial microRNA that alters the expression level of the target nucleic acid.

[0358] Effective knockdown of a target can be determined by methods in the art, and will exhibit minimal, if any, off-target effects.

[0359] When measuring processing, effective passenger-guide strand duplexes of regulatory polynucleotides (eg, prim- or pre-microRNAs) exhibit guide to passenger strand ratios greater than 95%.

[0360] Example 2. Passenger-Guidance Chain Optimization

[0361] To achieve specific and efficient targeted knockdown or modulation of target expression, the passenger and guide strands that will form the duplex stem of the stem-loop structure of the primary- or pre-microRNA of the invention can be optimized, respectively, for example as siRNA (small interfering RNA).

[0362] siRNAs are designed against a selected target nucleic acid as classical siRNAs with a 19 base pair central duplex with a 3' dinucleotide overhang on the 3' end of the strands of the duplex and wherein the antisense strand has perfect complementarity to the target nucleic acid over a 19 nucleotide region.

[0363] Alternatively, siRNAs are designed in which the sense strand (passenger strand) contains less than 19 nucleotides of identity to the target nucleic acid.

[0364] Modifications to the sense-antisense (passenger-guide) strand duplex base pairing are made to introduce bulges or mismatches. Insertions or deletions or mismatches may be introduced at the 5' or 3' end of the sense strand, and these insertions or deletions may or may not be reflected on the guide strand.

[0365] The resulting siRNAs are tested for target knockdown and other relevant physiological and pharmacokinetic properties, and for the extent of off-target effects by standard methods known in the art.

[0366] siRNAs that exhibit adequate target knockdown and minimal off-target effects (with or without further modification) are then engineered as passenger and guide strands of the primary- or pre-microRNAs of the invention.

[0367] Example 3. Design of passenger-guide strand for SOD1

[0368]

[0369] The 19-mer is shown in Table 4 together with the most 5' position of the sense strand, along with the antisense strand which is the reverse complement of the sense strand.

[0370] The 19-mer served as the core starting sequence for designing the siRNAs to be tested.

[0371] Table 4. SOD1 19-mer

[0372]

[0373]

[0374]

[0375]

[0376]

[0377]

[0378]

[0379] The core starting sense-antisense pairs of Table 4 above were then engineered into duplex siRNAs. In doing so, the 3'-most nucleotide of the sense strand was changed to a cytidine (C) nucleotide in all cases, leaving only 18 nucleotides with identity to the target.

[0380] Then, dinucleotide termini at the 3' end of each of the sense and antisense strands were added, thereby generating the duplexes of Table 5.

[0381] Table 5. siRNA duplexes targeting SOD1

[0382]

[0383]

[0384]

[0385]

[0386]

[0387]

[0388]

[0389]

[0390]

[0391]

[0392]

[0393]

[0394]

[0395] The siRNAs were then annealed and tested for SOD1 knockdown.

[0396] Example 4. Primary- and pre-microRNAs targeting SOD1

[0397] The passenger-guide strand duplex of the SOD1 siRNA found to be effective from the experiments in Example 3 was engineered into an expression vector and transfected into cells of the central nervous system or neuronal cell line. Even though the overhangs used in the siRNA knockdown studies were the classic dTdT for siRNA, the overhangs in the synthetic primary-miR or pre-miR can contain any dinucleotide overhangs.

[0398] The cells used may be primary cells or derived from induced pluripotent stem cells (iPS cells).

[0399] SOD1 knockdown was then measured and deep sequencing was performed to determine the exact passenger and guide strands processed from each primary- or pre-microRNA administered in the expression vector.

[0400] The guide to passenger strand ratio is calculated to determine the efficiency of the knockdown, e.g., the efficiency of the knockdown of RNA-induced silencing complex (RISC) processing.

[0401] The N-terminus was sequenced to identify the cleavage site and to determine the uniform cleavage percentage of the target. As expected, cleavage was above 90%.

[0402] In a parallel study HeLa cells were co-transfected to analyze in vitro knockdown of SOD1. Luciferase constructs were used as controls to determine off-target effects.

[0403] Deep sequencing was performed again.

[0404] Example 5. Primary- and pre-microRNAs targeting SOD1

[0405] According to the present invention, primary- and pre-microRNAs are designed. These are given in Tables 6A, 6B, 7A and 7B. The sequences are described in 5' to 3' directions, and the region of the stem-loop structure is opened in this order in the table. In Tables 7A and 7B, the "miR" component of the name of the sequence does not necessarily correspond to the sequence number of the miRNA gene (for example, VOYmiR-101 is the name of the sequence, and does not necessarily mean that miR-101 is part of the sequence).

[0406] Table 6A. Pre-miR sequences (5'-3')

[0407]

[0408]

[0409]

[0410] Table 6B. Pre-miR sequences (5'-3')

[0411]

[0412] Table 7A. Primary-miR sequences (5'-3')

[0413]

[0414]

[0415]

[0416]

[0417]

[0418] Table 7B. Primary-miR sequences (5'-3')

[0419]

[0420] Example 6. Primary- and pre-microRNAs targeting SOD1; in vivo studies In vivo studies were performed to test the efficacy of the primary- or pre-microRNA constructs of Example 5.

[0421] Table 8 summarizes the experimental design variables to be explored.

[0422] The designs of regulatory nucleic acids (primary- or pre-microRNAs) included a loop region derived from miR30, a stem region derived from let7 and different combinations of passenger strands varying in bulges, mismatches and asymmetric regions.

[0423] Table 8. Experimental design

[0424]

[0425] Example 7. Primary-miRNA constructs in AAV-miRNA vectors

[0426] The passenger-guide strand duplexes of the SOD1 siRNA listed in Table 7 were engineered into AAV-miRNA expression vectors. The constructs (listed from ITR to ITR, 5' to 3') contained mutant ITRs, promoters (CMV, U6 or CB6 promoters (which include the CMVie enhancer, CBA promoter and SV40 intron), the primary-miRNA constructs from Table 7, rabbit globin polyadenylation, and wild-type ITRs. In vitro and in vivo studies were performed to test the efficacy of the AAV-miRNA expression vectors.

[0427] Example 8. Activity of primary-miRNA constructs in HeLa cells

[0428] The seven primary-miRNA constructs described in Example 7 (VOYmiR-103, VOYmiR-105, VOYmiR-108, VOYmiR-114, VOYmiR-119, VOYmiR-120, and VOYmiR-127) and a double-stranded mCherry control were transfected in HeLa cells to test the activity of the constructs.

[0429] A. Passenger and guide strand activity

[0430] 7 kinds of primary-miRNA constructs and double-stranded mCherry control transfection into HeLa cells.After 48 hours, endogenous mRNA expression was evaluated.All 7 kinds of primary-miRNA constructs showed high activity of guide strand (with 75-80% knockdown) and low to no activity of passenger strand.The guide strand of primary-miRNA candidate vector showed high activity, producing 75-80% SOD1 knockdown, while the passenger strand showed little to no activity.

[0431] B. Activity of miRNA on SOD1

[0432] Seven primary-miRNA constructs and a double-stranded mCherry control (dsmCherry) were transfected into HeLa cells at an MOI of 1e4 vg / cell, 1e3 vg / cell, or 1e2 vg / cell. After 72 hours, endogenous mRNA expression was evaluated. All seven primary-miRNA constructs showed effective knockdown at 1e3 vg / cell. Most primary-miRNA constructs showed high activity (75-80% knockdown), as shown in Figure 1 as shown in .

[0433] Example 9. Activity of primary-miRNA constructs

[0434] The 30 primary-miRNA constructs described in Example 7 (VOYmiR-102.860, VOYmiR-102.861, VOYmiR-102.866, VOYmiR-102.870, VOYmiR-102.823, VOYmiR-104.860, VOYmiR-104.861, VOYmiR-104.866, VOYmiR-104.870, VOYmiR-104.823, VOYmiR-109.860, VOYmiR-109.861, VOYmiR-109.866, VOYmiR-109.870, VOYmiR-109.823, VOYmiR-11 4.860, VOYmiR-114.861, VOYmiR-114.866, VOYmiR-114.870, VOYmiR-114.823, VOYmiR-116.860, VOYmiR-116.861, VOYmiR-116.866, VOYmiR-116.870, VOYmiR-116.823, VOYmiR-127.860, VOYmiR-127.861, VOYmiR-127.866, VOYmiR-127.870, VOYmiR-127.823) as well as VOYmiR-114 and double-stranded mCherry controls were transfected into cells to test the activity of the constructs.

[0435] A. Activity of passenger and guide strands in HEK293

[0436] Thirty primary-miRNA constructs and two controls were transfected into HEK293T cells. After 24 hours, endogenous mRNA expression was evaluated. Most primary-mRNA constructs showed high activity of the guide strand ( Figure 3 ) and low to no activity of the passenger strand ( Figure 4 ).

[0437] B. Activity of passenger and guide strands in HeLa

[0438] Thirty primary-miRNA constructs and two controls were transfected into HeLa cells. After 48 hours, endogenous mRNA expression was evaluated. Most primary-mRNA constructs showed high activity of the guide strand ( Figure 5 ) and low to no activity of the passenger strand ( Figure 6 ).

[0439] C. Correlation between HeLa and HEK293

[0440] Knockdown of the 30 primary-miRNAs was similar between HeLa and HEK293 cells. The 30 primary-miRNA constructs showed knockdown of the guide strand of the construct (see Figure 3 and Figure 5 ). Most guide strands of the primary-miRNA constructs showed 70-90% knockdown.

[0441] D. Capsid Selection

[0442] The top primary-miRNA constructs from HeLa and HEK293 were packaged into AAV and subjected to HeLa infection. To determine the best AAV to package the constructs, mCherry packaged in AAV2 or AAV-DJ8 was infected into HeLa cells at an MOI of 10 vg / cell, 1e2 vg / cell, 1e3 vg / cell, 1e4 vg / cell or 1e5 vg / cell and expression was evaluated at 40 hours. AAV2 was selected as the capsid to package the top primary-miR constructs.

[0443] E. AAV2 production

[0444] The leading primary-miRNA constructs from HeLa and HEK293 were packaged into AAV2 (1.6 kb), and a double-stranded mCherry control (dsmCherry) was also packaged. The packaged constructs were subjected to Idoixanol purification before analysis. AAV titers are shown in Table 9.

[0445] Table 9. AAV titers

[0446]

[0447]

[0448] The effect of transduction on SOD1 knockdown in HeLa cells was shown in Figure 7 Additionally, in HeLa cells, a larger MOI (1.0E+04 vs. 1.0E+05) did not show increased knockdown for either construct.

[0449] F. Activity of constructs in human motor neuron progenitors (HMNPs)

[0450] The first 18 primary-miRNA constructs described in Example 9E and a control for mCherry were infected into human motor neuron progenitor (HMNP) cells at an MOI of 10E5. After 48 hours, endogenous mRNA expression was evaluated. About half of the constructs gave greater than 50% silencing of SOD1 in HMNPs, and 4 of them gave greater than 70% silencing ( Figure 8 ).

[0451] G. Construct selection for in vivo studies

[0452] The top 12 primary-miRNA packaged constructs were selected that had major effects on the target sequence and minor effects on the cassette. These constructs packaged in AAV-rh10 capsids were formulated as injections and administered in mammals to study the in vivo effects of the constructs.

[0453] H. Activity in different cell lines

[0454] The activity of the primary-miRNA packaged constructs was tested in HeLa, SH-SY5Y, U87MG and primary human astrocyte cells. The activity in HeLa cells was in the range of 1-5 pM. The activity in SH-SY5Y cells was in the range of 13-17 pM. The activity in U87MG cells was about 1 pM. The activity in primary human astrocytes was in the range of 49-123 pM.

[0455] Example 10. In vitro studies of primary-miRNA

[0456] The 18 primary-miRNAs and mCherry controls described in Example 9D packaged in AAV2 were used for this study. For this study, the cells were incubated in culture medium (500 ml DMEM / F-12 GLUTAMAX TM HEK293T cells (Fisher Scientific, catalog number HCL4517) in culture medium (500 ml DMEM / F-12GLUTAMAX) supplement (Life Technologies, catalog number 10565-018), 50 ml FBS (Life Technologies, catalog number 16000-044, lot number: 1347556), 5 ml MEM non-essential amino acid solution (100x) (catalog number 11140-050) and 5 ml HEPES (1M) (Life Technologies, catalog number 15630-080)). TMSupplement (Life Technologies, catalog number 10565-018), 50ml FBS (Life Technologies, catalog number 16000-044, batch number: 1347556), 5ml MEM non-essential amino acids solution (100x) (catalog number 11140-050) and 5ml HEPES (1M) (Life Technologies, catalog number 15630-080)) or normal human astrocytes (HA) (Lonza, catalog number CC-2565) in culture medium (ABM basal medium 500ml (Lonza, catalog number CC-3186) supplemented with AGM SingleQuotKit Suppl. & Growth Factors (Lonza, catalog number CC-4123)) were used to test the constructs. HEK293T cells (5x10E4 cells / well in 96-well plate), U251MG cells (2x10E4 cells / well in 96-well plate) and HA cells (2x10E4 cells / well in 96-well plate) were seeded, and the MOI used to infect the cells was 1.0E+05. After 48 hours, the cells were analyzed and the results are shown in Table 10.

[0457] Table 10. Relative SOD1 mRNA levels

[0458]

[0459]

[0460] For most constructs, greater than 80% knockdown was seen in HEK293T cells. More than half of the constructs showed greater than 80% knockdown in U251MG cells and in HA cells.

[0461] Example 11. Dose-dependent reduction of SOD1

[0462] Four of the first 18 primary-miRNA constructs described in Example 9E and a control of mCherry were transfected into a human astrocyte cell line (U251MG) or primary human astrocytes (HA) at an MOI of 1.0E+02, 1.0E+03, 1.0E+04, 1.0E+05, or 1.0E+06. After 48 hours, endogenous mRNA expression and dose-dependent silencing were evaluated and shown in Fig. 9 (U251MG) and Fig.10 For all constructs, increasing doses were also associated with increasing amounts of knocked-down SOD1 mRNA.

[0463] Example 12. Time course of SOD1 knockdown

[0464] Two primary-miRNA constructs (VOYmiR-120 and VOYmiR-122), negative control and positive control of SOD1 siRNA were transfected into a human astrocyte cell line (U251MG). Relative SOD1 mRNA was determined for 60 hours, as in Fig.11 As shown in . Following Nucleofector transfection, 70-75% knockdown of hSOD1 was seen for both primary-miR constructs, with maximal knockdown seen in the 12-24 hour window.

[0465] Example 13. SOD1 knockdown and standing percentage

[0466] VOYmiR-104 was transfected into HeLa cells at concentrations of 50 pM, 100 pM, and 150 pM and compared to untreated (UT) cells. Relative SOD1 mRNA, percentage of guide strand, and percentage of passenger strand were determined at 36, 72, 108, and 144 hours, as shown in Figures 12A-12C The highest concentration (150 pM) showed the greatest reduction in expression, but all three doses showed a significant reduction in SOD1 expression.

[0467] Example 14. Primary-miRNA targeting SOD1

[0468] Primary-miRNAs were designed for Dog SOD1 and the constructs are given in Table 11. Dog SOD1 is 100% conserved with humans in the regions targeted by the present invention. The sequences are described in the 5' to 3' direction and the regions of the stem-loop structure are opened in this order in the table. In Table 11, the "miR" component of the name of the sequence does not necessarily correspond to the sequence number of the miRNA gene (e.g., dVOYmiR-102 is the name of the sequence and does not necessarily mean that miR-102 is part of the sequence).

[0469] Table 11. Dog primary-miR sequences (5'-3')

[0470]

[0471]

[0472] Example 15. Effect of the Position of Regulatory Polynucleotides

[0473] A. Effect on Virus Titer

[0474] The regulatory polynucleotides (VOYmiR-114 or VOYmiR-126) were inserted into the expression vector (genome size approximately 2400 nucleotides; scAAV) at 6 different locations, as shown in Fig.13 As shown in Fig.13 In the , "ITR" is an inverted terminal repeat, "I" represents an intron, "P" is polyadenylation and "MP" is a regulatory polynucleotide. For the 6 positions and for the control (construct without a regulatory polynucleotide; scAAV), the viral titer was evaluated using TaqMan PCR and the results are shown in Table 12.

[0475] Table 12. Virus titers

[0476]

[0477]

[0478] B. Impact on genome integrity

[0479] The regulatory polynucleotide (VOYmiR-114) was inserted into an expression vector (genome size 2400 nucleotides; scAAV) and a control without the regulatory polynucleotide (scAAV) at 6 different locations, as shown in Fig.13 As shown in Fig.13 In the , "ITR" is an inverted terminal repeat, "I" represents an intron, "P" is polyadenylation and "MP" is a regulatory polynucleotide. Viral genomes were extracted from purified AAV preparations and run on neutral agarose gels. Truncated genomes were seen in all constructs, and the approximate percentage of truncated genomes (percent of total) is shown in Table 13.

[0480] Table 13. Truncated genomes

[0481] Constructs % of total Position 1 50 Position 2 42 Location 3 49 Position 4 34 Location 5 33 Position 6 59 Comparison 9

[0482] Position 6 had the largest number of truncated genomes, while positions 4 and 5 had the smallest amounts of truncated genomes.

[0483] C. Effect on knockdown efficiency

[0484] The regulatory polynucleotide (VOYmiR-114) was inserted into an expression vector (AAV2) (genome size 2400 nucleotides; scAAV) at 6 different locations, as in Fig.13 As shown in Fig.13 In the , "ITR" is an inverted terminal repeat, "I" stands for an intron, "P" is polyadenylation and "MP" is a regulatory polynucleotide. 4 vg / cell, 1x 10 3vg / cell and 1x10 2 vg / cell for transduction of HeLa cells. SOD1 mRNA expression was determined 72 hours after infection as a percentage of control (eGFP) and the results are shown in Table 14.

[0485] Table 14. SOD1 expression

[0486]

[0487] Position 3 had the highest SOD1 mRNA expression (as % of control), and position 4 had the lowest SOD1 mRNA expression (as % of control).

[0488] Example 16. Effect of genome size

[0489] A. Effect on Virus Titer

[0490] The regulatory polynucleotide (VOYmiR-114) was inserted into the expression vector (genome size 2 kb; scAAV) at positions 1, 2, 5 and 6, as in Fig.13 As shown in Fig.13 In the table, "ITR" is an inverted terminal repeat, "I" represents an intron, "P" is a polyadenylic acid and "MP" is a regulatory polynucleotide. A double-stranded control without a regulatory polynucleotide (genome size 1.6 kb; scAAV control) and a double-stranded expression vector (scAAV miR114; ITR (105 nucleotides) - promoter (~900 nucleotides) - regulatory polynucleotide (158 nucleotides) - polyadenylic acid sequence (127 nucleotides) and ITR) were compared, as well as a control without a regulatory polynucleotide (eGFP; scAAV). Viral titers were evaluated using TaqMan PCR, and the results are shown in Table 15.

[0491] Table 15. Virus titers

[0492] Construct size Virus titer (VG / 15-cm dish) Position 1 2kb 9.5E+10 Position 2 2kb 1.2E+11 scAAV miR114 1.6kb 1.1E+11 Location 5 2kb 2.4E+10 Position 6 2kb 1.1E+11 Comparison 2kb 2.2E+11

[0493] The lowest viral titers were seen with the position 5 construct and the highest with the position 2 construct.

[0494] B. Impact on genome integrity

[0495] The regulatory polynucleotide (VOYmiR-114) was inserted into the expression vector (genome size 2 kb; scAAV) at positions 1, 2, 5 and 6, as in Fig.13 As shown in Fig.13In, "ITR" is an inverted terminal repeat, "I" represents an intron, "P" is polyadenylation and "MP" is a regulatory polynucleotide. The double-stranded control without regulatory polynucleotides (genome size 1.6kb; scAAV control) and double-stranded expression vectors (scAAV miR114; ITR (105 nucleotides)-promoter (~900 nucleotides)-regulatory polynucleotides (158 nucleotides)-polyadenylation sequence (127 nucleotides) and ITR) were compared, as well as controls without regulatory polynucleotides (eGFP; scAAV). Truncated genomes were seen in all constructs, and the approximate percentages of truncated genomes (percentage of the total) are shown in Table 16.

[0496] Table 16. Truncated genomes

[0497] Constructs size % of total Position 1 2kb 34 Position 2 2kb 30 scAAV miR114 1.6kb 20 Location 5 2kb 21 Position 6 2kb 46 Comparison 2kb 5

[0498] All constructs were determined to have some truncated genomes.

[0499] Another study was performed to determine the effects of different regulatory polynucleotides. VOYmiR-114 and VOYmiR-126 were inserted into separate expression vectors (genome size 1.6 kb; scAAV) with the regulatory polynucleotides close to the 3' ITR (forward orientation). For the VOYmiR-114 construct, the distance between the 5' end of the vector genome (1526 nucleotides) and the center of the regulatory polynucleotide (the center of the flexible loop) was 1115 nucleotides. For the VOYmiR-126 construct, the distance between the 5' end of the vector genome (1626 nucleotides) and the center of the regulatory polynucleotide (the center of the flexible loop) was 1164 nucleotides.

[0500] For the VOYmiR-114 construct, the viral titer (VG / 15-cm dish) was about 1.1E+11. For the VOYmiR-126 construct, the intron probe viral titer (VG / 15-cm dish) was about 1.2E+12. The control was about 2.1E+11 (VG / 15-cm dish). VOYmir-114 had a genome that was about 20% truncated, VOYmiR-126 had a genome that was about 15% truncated, and the control had a genome that was about 5% truncated.

[0501] Example 17. Effect of single-chain constructs

[0502] A. Effect on Virus Titer

[0503] The regulatory polynucleotide (VOYmiR-114) was inserted into the expression vector (genome size 4.7 kb; ssAAV) at positions 1, 3, and 5, as in Fig.13As shown in , a control without regulatory polynucleotides was also tested (genome size 2 kb; ssAAV). Fig.13 In the expression vector, "ITR" is an inverted terminal repeat, "I" represents an intron, "P" is polyadenylation and "MP" is a regulatory polynucleotide. Virus titer was evaluated using TaqMan PCR and the results are shown in Table 17.

[0504] Table 17. Virus titers

[0505] Construct Virus titer (VG / 15-cm dish) Position 1 5.0E+11 Location 3 7.5E+11 Location 5 3.5E+11 Comparison 2.5E+11

[0506] Position 3 showed the greatest viral titer, followed by position 1 and then position 5.

[0507] B. Impact on genome integrity

[0508] The regulatory polynucleotide (VOYmiR-114) was inserted into the expression vector (genome size 4.7 kb; ssAAV) at positions 1, 3, and 5, as in Fig.13 As shown in , a control without regulatory polynucleotides was also tested (genome size 2 kb; ssAAV). Fig.13 In the table 18, "ITR" is an inverted terminal repeat, "I" represents an intron, "P" is a polyadenylate and "MP" is a regulatory polynucleotide. Viral genomes were extracted from purified AAV preparations and run on neutral agarose gels. Truncated genomes were seen in all constructs, and the approximate percentage of truncated genomes (percent of the total) is shown in Table 18.

[0509] Table 18. Truncated genomes

[0510] Constructs % of total Position 1 48 Location 3 30 Location 5 72 Comparison 0

[0511] Position 5 had the largest number of truncated genomes, while position 3 had the smallest amount of truncated genomes.

[0512] C. Effect on knockdown efficiency

[0513] The regulatory polynucleotide (VOYmiR-114) was inserted into the expression vector (genome size 4.7 kb; ssAAV) at positions 1, 3, and 5, as in Fig.13 As shown in , there are single-stranded controls without regulatory polynucleotides (genome size 2 kb; ssAAV control), double-stranded controls without regulatory polynucleotides (genome size 1.6 kb; scAAV control) and double-stranded expression vectors (scAAV miR114; ITR (105 nucleotides) - promoter (~900 nucleotides) - regulatory polynucleotides (158 nucleotides) - poly (A) sequence (127 nucleotides) and ITRs). Fig.13 In the , "ITR" is an inverted terminal repeat, "I" stands for an intron, "P" is polyadenylation and "MP" is a regulatory polynucleotide. 4 vg / cell, 1x 10 3 vg / cell and 1x10 2 The transduction of HeLa cells was performed with 100 vg / cell. SOD1 mRNA expression was determined 72 hours after infection as a percentage of the control (eGFP) and the results are shown in Table 19.

[0514] Table 19. SOD1 expression

[0515]

[0516]

[0517] Position 3 had the highest SOD1 mRNA expression (as % of control), followed by position 1, and the single-stranded construct with the lowest SOD1 mRNA expression (as % of control) was position 5. None of the single-stranded constructs had as low a knockdown efficiency as the double-stranded control with the regulatory polynucleotide.

[0518] Example 18. SOD1 knockdown in vivo

[0519] To evaluate the in vivo biological activity of primary-miRNAs, self-complementary primary-miRNAs (VOYmiR-114.806, VOYmiR127.806, VOYmiR102.860, VOYmiR109.860, VOYmiR114.860, VOYmiR116.860, VOYmiR127.860, VOYmiR102.861, VOYmiR104.861, VOYmiR109.861, VOYmiR114.861, VOYmiR109.866, VOYmiR116.866, or VOYmiR127.866) were packaged in AAV-DJ with a CBA promoter.

[0520] In mice, these packaged primary-miRNAs or vehicle-only controls (phosphate buffered saline containing 5% sorbitol and 0.001% F-68) were administered by 10-minute intrastriatal infusion. Female or male Tg(SOD1)3Cje / J mice (Jackson Laboratory, Bar Harbor, ME) expressing human SOD1 and having a body weight of approximately 20-30 g received a unilateral injection of 5 uL of the test substance targeted to the striatum (anteroposterior + 0.5 mm, mediolateral + 2 mm, relative to the bregma; dorsal ventral 3.8 mm, relative to the surface of the skull). The test substance was injected at 0.5 uL / min (5 animals per test substance). A Hamilton microsyringe (1701 type, 10 μl) with a pre-filled pump regulation with a 33-gauge needle was used. After injection 1,2,3,4 or 6 weeks, animals were killed, brains were taken out, and the ipsilateral striatum (including the infusion site) from 1mm coronal slab and the striatal tissue from adjacent 1mm coronal slab was cut and quickly frozen. Mouse tissue samples were cracked, and human SOD1 protein levels and SOD1 and mouse GAPDH (mGAPDH) mRNA levels were quantitatively analyzed. SOD1 protein levels were quantitatively analyzed by ELISA (eBioscience (Affymetrix, San Diego, CA)), and total protein levels were quantitatively analyzed by BCA (ThermoFisher Scientific, Waltham, MA). For each tissue sample, the SOD1 protein level normalized to total protein was calculated as the mean value of 2 determinations. These normalized SOD1 protein levels were further normalized to the vehicle group, and then averaged to obtain the group (treatment) mean value. SOD1 and mGAPDH mRNA levels were quantitatively analyzed by qRT-PCR. For each tissue sample, the ratio of SOD1 / mGAPDH (normalized SOD1 mRNA level) was calculated as the average of 3 determinations. These ratios were then averaged to obtain group (treatment) means. These group means were further normalized to the vehicle group.

[0521] In nonhuman primates, test articles (1 x 10 13 -3x 10 13vg of primary-miRNA packaged in AAV-DJ with CBA promoter) or vehicle. Female cynomolgus monkeys (bundled monkeys, CR Research Model Houston, Houston, TX) with a body weight of about 2.5-8.5 kg received a single intrathecal catheter implanted, the tip of the catheter being located at the lumbar vertebrae. The test article (4 animals per test article) was administered at intervals of about 60 minutes, including 3 1 mL rapid push injections (1 mL / minute). 4-6 weeks after administration, the animals were sacrificed, and the selected tissues were harvested for bioanalysis and histological evaluation. For inhibition after treatment with primary-miRNA packaged in AAV-DJ with CBA promoter, SOD1 protein and mRNA levels were assessed relative to the vehicle group.

[0522] Example 19. In vivo SOD1 knockdown using VOYmiR-114.806

[0523] In Tg(SOD1)3Cje / J mice, VOYmiR-114.806 packaged in AAVDJ with CBA promoter was administered as described in Example 18. Mice were administered 3.7 x 10 9 There was no significant decrease in normalized SOD1 protein levels after 1 or 2 weeks; normalized SOD1 protein levels were 98% and 10% of the vehicle control group after 1 and 2 weeks, respectively. + 11% (standard deviation) and 98 + By week 3, VOYmiR-114.806 reduced normalized SOD1 protein levels to 84% of the vehicle control group. + 9.0%, which was statistically significant (p<0.05, one-way ANOVA with Dunnett's post hoc analysis). By weeks 4 and 6, VOYmiR-114.806 reduced normalized SOD1 protein levels to 73% and 10% of the vehicle control group, respectively. + 7.9% (p<0.0001) and 75 + 7.4% (p<0.0001). These results confirm that VOYmiR-114.806 packaged in AAV-DJ with CBA promoter is effective in downregulating SOD1 protein levels in vivo. In addition, these results confirm that as low as 3.7 x 10 9 Total intrastriatal dose of VOYmiR-114.806 packaged in AAVDJ with CBA promoter resulted in significant downregulation of SOD1 protein levels.

[0524] Although the present invention has been described with considerable detail and with considerable particularity with respect to several described embodiments, it is not intended that the present invention be limited to any such detail or embodiment or to any particular embodiment, but rather the present invention should be construed with reference to the appended claims, thereby providing the broadest possible interpretation of such claims in view of the prior art and thereby effectively encompassing the intended scope of the present invention.

[0525] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict, the present specification and the definitions therein shall prevail. In addition, the section headings, materials, methods, and examples are merely exemplary and are not intended to be limiting.

Claims

1. An adeno-associated virus genome comprising a nucleotide sequence placed between two inverted terminal repeats, wherein the nucleotide sequence encodes a regulatory polynucleotide, wherein the regulatory polynucleotide comprises (a) a first flanking region comprising a nucleotide sequence of any one of SEQ ID NOs: 1-4; (b) a loop region comprising the nucleotide sequence of any one of SEQ ID NOs: 5-9; and (c) a second flanking region comprising the nucleotide sequence of any one of SEQ ID NOs: 10-14 or 810.

2. The adeno-associated virus genome according to claim 1, wherein the regulatory polynucleotide further comprises a guide strand and a passenger strand, optionally wherein: (i) the passenger strand is located between the first flanking region and the loop region and the guide strand is located between the loop region and the second flanking region; (ii) the guide strand is located between the first flanking region and the loop region and the passenger strand is located between the loop region and the second flanking region; (iii) the guide strand, the passenger strand, or both are 19-22 nucleotides in length; and / or (iv) the guide strand is at least 70% complementary to a target RNA, wherein the target RNA is expressed in a nervous system cell, tissue or organ.

3. The adeno-associated virus genome according to claim 1, further comprising: (i) a promoter operably linked to a nucleic acid encoding a regulatory polynucleotide; (ii) enhancers; (iii) intronic regions; and / or (iv) Polyadenylation signal region.

4. The adeno-associated virus genome according to claim 3, wherein: (i) the promoter is chicken β-actin, elongation factor 1 α-subunit promoter, synapsin promoter, methyl-CpG binding protein 2 promoter, glial fibrillary acidic protein promoter, neuron-specific enolase promoter, myelin basic protein promoter, PGK promoter, CAG promoter, ubiquitin C promoter or cytomegalovirus promoter; (ii) the enhancer comprises a cytomegalovirus enhancer; and / or (iii) The intron comprises the SV40 intron.

5. A recombinant adeno-associated virus, comprising the adeno-associated virus genome according to any one of claims 1 to 4 and an AAV capsid, optionally wherein the AAV capsid comprises an AAV5 capsid or a variant thereof, or an AAV9 capsid or a variant thereof. A vector comprising the adeno-associated virus genome according to any one of claims 1 to 4.

7. A cell comprising the adeno-associated virus genome according to any one of claims 1 to 4, optionally wherein the cell is a HEK239 cell, a cell of the central nervous system, a motor neuron or an astrocyte.

8. A pharmaceutical composition comprising the recombinant adeno-associated virus according to claim 5, and a pharmaceutically acceptable excipient.

9. Use of the recombinant adeno-associated virus according to claim 5 in the preparation of a medicament for treating a disease of the central nervous system in a subject.

10. The use according to claim 9, wherein: (i) the recombinant adeno-associated virus is formulated for intravenous administration, administration via intracisternal injection, intravascular administration and / or intraventricular administration; and / or (ii) The disease of the central nervous system is Huntington's disease or amyotrophic lateral sclerosis.

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