Amyloid precursor protein (APP) RNAi reagents

Abstract

Provided herein are APP RNAi agents, and compositions comprising APP RNAi agents. Also provided herein are methods of using an APP RNAi agent or a composition comprising an APP RNAi agent in reducing APP expression and / or treating APP-related neurological diseases.

Description

[0001] sequence list This application is submitted together with a sequence list in ST.26 XML format. The sequence list is provided as a file named "30578_WO" created on June 14, 2024, and is 1.576 megabytes in size. The sequence list information in ST.26 XML format is incorporated herein by reference in its entirety. Background of the Invention Amyloid precursor protein (APP) is a transmembrane protein expressed in neurons and glial cells. APP is cleaved by β-secretase and γ-secretase to release β-amyloid (Aβ) peptides, a group of peptides comprising 38-43 amino acid residues. Aβ monomers aggregate into various types of higher-order structures, including oligomers, fibrils, and amyloid filaments. Amyloid oligomers are soluble and can diffuse throughout the brain, while amyloid filaments are larger and insoluble and can further aggregate to form amyloid deposits or plaques. Amyloid plaques in the brain have been associated with a variety of conditions and diseases, including Alzheimer's disease (AD), Down syndrome, and cerebral amyloid angiopathy (CAA).

[0003] The blood-brain barrier (BBB) ​​is a selectively semi-permeable boundary formed by capillary endothelial cells that prevents solutes, including pathogens, from entering the central nervous system (CNS). The BBB allows some small molecules to pass through by passive diffusion, and BBB cells use specific transport proteins to actively transport metabolites essential for neurological function, such as glucose and amino acids, across the barrier. The BBB has a neuroprotective function by tightly controlling access to the brain; however, it also hinders the access of therapeutic agents to the CNS. Antibodies targeting the transferrin receptor (“TfR”) have been used to modulate BBB transport. However, attempts to shuttle therapeutic agents across the BBB using anti-TfR antibodies have proven challenging. To date, no TfR shuttles or conjugates have been approved for the treatment of CNS diseases.

[0004] RNA interference (RNAi) is a highly conserved regulatory mechanism in which RNA molecules are involved in gene expression through sequence-specific inhibition of double-stranded RNA molecules (dsRNA) (Fire et al., Nature 391:806-811, 1998).

[0005] Currently, there are no disease-modifying therapies available for Down syndrome and cerebral amyloid angiopathy. Although the FDA recently approved two anti-Aβ antibodies (adecanumab and lencanemab) for the treatment of Alzheimer's disease (AD), AD patients vary widely in terms of disease progression, symptom onset, the trajectory of cognitive and functional decline, and their response to treatment. Therefore, there remains a need for therapeutic agents that can cross the BBB and reach the CNS, and attack the initiation of the amyloid cascade, for example, by utilizing RNAi to inhibit APP mRNA expression, thereby reducing the production and / or levels of pathogenic Aβ peptides. Invention Overview This article provides APP RNAi reagents that can access the central nervous system (CNS) and reduce APP mRNA expression, as well as compositions containing APP RNAi reagents. This article also provides methods for using APP RNAi reagents or compositions containing APP RNAi reagents to reduce APP expression and / or treat APP-related neurological disorders.

[0007] In one respect, this paper provides inclusion equation (I): (RL) n -P is an APP RNAi reagent, wherein R is a double-stranded RNA (dsRNA) containing a sense strand and an antisense strand, and said antisense strand is complementary to APP mRNA; wherein P is a protein containing a monovalent human TfR-binding domain (“human TfR-binding protein”); wherein L is a linker, or optionally absent; and wherein n is an integer from 1 to 3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3.

[0008] In some implementations, this document provides inclusion formula (I): (RL) n-P is an APP RNAi reagent, wherein R is a double-stranded RNA (dsRNA) containing a sense strand and an antisense strand, and said antisense strand is complementary to APP mRNA; wherein P is a protein containing a monovalent human TfR binding domain; wherein L is a linker, or optionally absent, wherein said human TfR binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein said VH comprises heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and said VL comprises light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 3, LCDR1 comprises SEQ ID NO: 4, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 6; and wherein n is an integer from 1 to 3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, VH comprises SEQ ID NO: 7, and VL comprises SEQ ID NO: 8. In some embodiments, VH comprises a sequence having at least 95% sequence identity with SEQ ID NO: 7, and VL comprises a sequence having at least 95% sequence identity with SEQ ID NO: 8. Exemplary sequences of human TfR binding domains and proteins are provided in Tables 1a and 1b.

[0009] In some implementations, L is a Mal-Tet-TCO connector, an SMCC connector, or a GDM connector (see Table 4). In some implementations, L is an SMCC connector as shown in Table 4.

[0010] In another aspect, this document provides APP RNAi reagents comprising double-stranded RNA (dsRNA) containing a sense strand and an antisense strand, wherein the sense and antisense strand sequences are selected from Tables 5a, 5b, 7a, and 7b. In some embodiments, the APP RNAi reagent comprises any dsRNA from Tables 5a, 5b, 7a, and 7b.

[0011] Tables 5a and 5b provide exemplary unmodified sense and antisense sequences of dsRNA targeting human APP mRNA. In some embodiments, the sense and antisense strands of the dsRNA comprise a pair of nucleic acid sequences selected from the following: (a) The sense chain contains SEQ ID NO: 35, and the antisense chain contains SEQ ID NO: 36; (b) The sense chain contains SEQ ID NO: 37, and the antisense chain contains SEQ ID NO: 38; (c) The sense chain contains SEQ ID NO: 39, and the antisense chain contains SEQ ID NO: 40; (d) The sense chain contains SEQ ID NO: 41, and the antisense chain contains SEQ ID NO: 42; (e) The sense chain contains SEQ ID NO: 43, and the antisense chain contains SEQ ID NO: 44; (f) The sense chain contains SEQ ID NO: 45, and the antisense chain contains SEQ ID NO: 46; (g) The sense chain contains SEQ ID NO: 47, and the antisense chain contains SEQ ID NO: 48; (h) The sense chain contains SEQ ID NO: 49, and the antisense chain contains SEQ ID NO: 50; (i) The sense chain contains SEQ ID NO: 51, and the antisense chain contains SEQ ID NO: 52; (j) The sense chain contains SEQ ID NO: 53, and the antisense chain contains SEQ ID NO: 54; (k) The sense chain contains SEQ ID NO: 55, and the antisense chain contains SEQ ID NO: 56; (l) The sense chain contains SEQ ID NO: 57, and the antisense chain contains SEQ ID NO: 58; (m) The sense chain contains SEQ ID NO: 59, and the antisense chain contains SEQ ID NO: 60; (n) The sense chain contains SEQ ID NO: 61, and the antisense chain contains SEQ ID NO: 62; (o) The sense chain contains SEQ ID NO: 63, and the antisense chain contains SEQ ID NO: 64; (p) The sense chain contains SEQ ID NO: 65, and the antisense chain contains SEQ ID NO: 66; (q) The sense chain contains SEQ ID NO: 67, and the antisense chain contains SEQ ID NO: 68; (r) The sense chain contains SEQ ID NO: 69, and the antisense chain contains SEQ ID NO: 70; (s) The sense chain contains SEQ ID NO: 71, and the antisense chain contains SEQ ID NO: 72; (t) The sense chain contains SEQ ID NO: 73, and the antisense chain contains SEQ ID NO: 74; (u) The sense chain contains SEQ ID NO: 75, and the antisense chain contains SEQ ID NO: 76; (v) The sense chain contains SEQ ID NO: 77, and the antisense chain contains SEQ ID NO: 78; (w) The sense chain contains SEQ ID NO: 79, and the antisense chain contains SEQ ID NO: 80; (x) The sense chain contains SEQ ID NO: 81, and the antisense chain contains SEQ ID NO: 82; (y) The sense chain contains SEQ ID NO: 83, and the antisense chain contains SEQ ID NO: 84; (z) The sense chain contains SEQ ID NO: 85, and the antisense chain contains SEQ ID NO: 86; (aa) The sense chain contains SEQ ID NO: 87, and the antisense chain contains SEQ ID NO: 88; (bb) The sense chain contains SEQ ID NO: 89, and the antisense chain contains SEQ ID NO: 90; (cc) The sense chain contains SEQ ID NO: 91, and the antisense chain contains SEQ ID NO: 92; (dd) The sense chain contains SEQ ID NO: 93, and the antisense chain contains SEQ ID NO: 94; (ee) The sense chain contains SEQ ID NO: 95, and the antisense chain contains SEQ ID NO: 96; (ff) The sense chain contains SEQ ID NO: 97, and the antisense chain contains SEQ ID NO: 98; (gg) The sense chain contains SEQ ID NO: 99, and the antisense chain contains SEQ ID NO: 100; (hh) The sense chain contains SEQ ID NO: 184, and the antisense chain contains SEQ ID NO: 36; (ii) The sense chain contains SEQ ID NO: 188, and the antisense chain contains SEQ ID NO: 38; and (jj) The sense chain contains SEQ ID NO: 35, and the antisense chain contains SEQ ID NO: 214; Optionally, one or more nucleotides of the sense and antisense strands are independently modified nucleotides, and the bonding between one or more nucleotides of the sense and antisense strands is a modified nucleotide bonding. In some embodiments, the sense strand comprises SEQ ID NO: 35, and the antisense strand comprises SEQ ID NO: 36. In some embodiments, the sense strand comprises SEQ ID NO: 35, and the antisense strand comprises SEQ ID NO: 214. In some embodiments, the sense strand comprises SEQ ID NO: 37, and the antisense strand comprises SEQ ID NO: 38.

[0012] dsRNA may include modifications. Modifications may be made to one or more nucleotides of the sense and / or antisense strands, or to internucleotide bonds. In some embodiments, one or more nucleotides of the sense and / or antisense strands are independently modified nucleotides, meaning that the sense and antisense strands may have different modified nucleotides. In some embodiments, each nucleotide of the sense strand is a modified nucleotide. In some embodiments, each nucleotide of the antisense strand is a modified nucleotide. In some embodiments, the modified nucleotide is a 2'-fluorinated nucleotide, a 2'-O-methylated nucleotide, a 2'-deoxynucleotide (DNA), or a 2'-O-alkylated nucleotide. In some embodiments, each nucleotide of the sense and antisense strands is an independently modified nucleotide, such as a 2'-fluorinated nucleotide, a 2'-O-methylated nucleotide, a 2'-deoxynucleotide (DNA), or a 2'-O-alkylated nucleotide.

[0013] In some embodiments, the sense strand has, for example, four 2'-fluorinated nucleotides at positions 7, 9, 10, and 11 from the 5' end of the sense strand. In some embodiments, at least one nucleotide of the sense strand is an unmodified RNA nucleotide. In some embodiments, at least one nucleotide of the sense strand is a 2' deoxyribonucleotide (DNA). In some embodiments, the other nucleotides of the sense strand are 2'-O-methylated nucleotides. In some embodiments, the antisense strand has, for example, four 2'-fluorinated nucleotides at positions 2, 6, 14, and 16 from the 5' end of the antisense strand. In some embodiments, the other nucleotides of the antisense strand are 2'-O-methylated nucleotides.

[0014] In some embodiments, the sense strand has, for example, three 2'-fluorinated nucleotides at positions 9, 10, and 11 from the 5' end of the sense strand. In some embodiments, at least one nucleotide of the sense strand is an unmodified RNA nucleotide. In some embodiments, at least one nucleotide of the sense strand is a 2' deoxyribonucleotide (DNA). In some embodiments, other nucleotides of the sense strand are 2'-O-methylated nucleotides. In some embodiments, the antisense strand has, for example, five 2'-fluorinated nucleotides at positions 2, 5, 7, 14, and 16 from the 5' end of the antisense strand. In some embodiments, the antisense strand has, for example, five 2'-fluorinated nucleotides at positions 2, 5, 8, 14, and 16 from the 5' end of the antisense strand. In some embodiments, the antisense strand has, for example, five 2'-fluorinated nucleotides at positions 2, 3, 7, 14, and 16 from the 5' end of the antisense strand. In some embodiments, other nucleotides of the antisense strand are 2'-O-methylated nucleotides.

[0015] In some implementations, the 5' end of the antisense chain has a phosphate analog, such as 5'-vinylphosphonate (5'-VP).

[0016] In some implementations, the sense or antisense chain includes a debasing moiety or a reverse debasing moiety.

[0017] In some embodiments, the sense strand and antisense strand have one or more modified nucleotide bonds. In some embodiments, the modified nucleotide bonds are phosphate thioester bonds. In some embodiments, the sense strand has four or five phosphate thioester bonds. In some embodiments, the antisense strand has four or five phosphate thioester bonds. In some embodiments, both the sense strand and antisense strand have four or five phosphate thioester bonds. In some embodiments, the sense strand has four phosphate thioester bonds, and the antisense strand has five phosphate thioester bonds.

[0018] Tables 7a and 7b provide exemplary sense and antisense strand sequences of modified dsRNA targeting human APP mRNA.

[0019] In some implementations, this document provides inclusion formula (I): (RL) n-P is an APP RNAi reagent, wherein R is a double-stranded RNA (dsRNA) containing a sense strand and an antisense strand, and said antisense strand is complementary to APP mRNA; wherein P is a protein containing a monovalent human TfR binding domain, and P is selected from TBP1, TBP2, TBP3, TBP4, or TBP5 in Table 1b; wherein L is a linker, or optionally absent, and wherein n is 1 or 3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, L is a linker in Table 4 (e.g., the SMCC linker in Table 4).

[0020] In some implementations, this document provides inclusion formula (I): (RL) n -P is an APP RNAi reagent, wherein R is a double-stranded RNA (dsRNA) containing sense and antisense strands, and the dsRNA is any dsRNA in Tables 5a, 5b, 7a, or 7b (e.g., dsRNA number 1); wherein P is a protein containing a monovalent human TfR binding domain; wherein L is a linker, or optionally absent; and wherein n is an integer from 1 to 3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, L is a linker in Table 4 (e.g., the SMCC linker in Table 4).

[0021] In some implementations, this document provides inclusion formula (I): (RL) n -P is an APP RNAi reagent, wherein R is a double-stranded RNA (dsRNA) containing sense and antisense strands, and the dsRNA is any dsRNA in Tables 5a, 5b, 7a, or 7b (e.g., dsRNA number 1); wherein P is a protein containing a monovalent human TfR binding domain, and P is selected from TBP1, TBP2, TBP3, TBP4, or TBP5 in Table 1b; wherein L is a linker, or optionally absent, and wherein n is an integer from 1 to 3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, L is a linker in Table 4 (e.g., the SMCC linker in Table 4).

[0022] In some implementations, this document provides inclusion formula (I): (RL) n-P is an APP RNAi reagent, wherein R is a double-stranded RNA (dsRNA) containing a sense strand and an antisense strand, and said antisense strand is complementary to APP mRNA; wherein P is a protein containing a monovalent human TfR-binding domain; wherein L is a linker, or optionally absent, wherein said human TfR-binding domain comprises two heavy chains HC1 and HC2 and one light chain LC1, wherein HC1 comprises SEQ ID NO: 14, LC1 comprises SEQ ID NO: 10, HC2 comprises SEQ ID NO: 15, and wherein n is 1 or 2. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, L is a linker from Table 4 (e.g., the SMCC linker from Table 4).

[0023] In some implementations, this document provides inclusion formula (I): (RL) n -P is an APP RNAi reagent, wherein R is a double-stranded RNA (dsRNA) containing a sense strand and an antisense strand, and said antisense strand is complementary to APP mRNA; wherein P is a protein containing a monovalent human TfR binding domain; wherein L is a linker, or optionally absent, wherein said human TfR binding domain comprises two heavy chains HC1 and HC2 and one light chain LC1, wherein HC1 comprises SEQ ID NO: 16, LC1 comprises SEQ ID NO: 10, HC2 comprises SEQ ID NO: 17, and wherein n is 1. In some embodiments, L is a linker from Table 4 (e.g., the SMCC linker from Table 4).

[0024] In some implementations, this document provides inclusion formula (I): (RL) n -P is an APP RNAi reagent, wherein R is a double-stranded RNA (dsRNA) containing a sense strand and an antisense strand, wherein the sense strand comprises SEQ ID NO: 35 and the antisense strand comprises SEQ ID NO: 36 or 214; wherein P is a protein containing a monovalent human TfR binding domain; wherein L is a linker, or optionally absent, and wherein n is an integer from 1 to 3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, L is a linker from Table 4 (e.g., the SMCC linker from Table 4).

[0025] In some implementations, this document provides inclusion formula (I): (RL) n-P is an APP RNAi reagent, wherein R is a double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand, wherein the sense strand comprises SEQ ID NO: 35 and the antisense strand comprises SEQ ID NO: 36 or 214; wherein P is a protein comprising a monovalent human TfR binding domain; wherein L is a linker, or optionally absent, wherein the human TfR binding domain comprises two heavy chains HC1 and HC2 and a light chain LC1, wherein HC1 comprises SEQ ID NO: 14, LC1 comprises SEQ ID NO: 10, HC2 comprises SEQ ID NO: 15, and wherein n is 1 or 2. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, L is a linker from Table 4 (e.g., the SMCC linker from Table 4).

[0026] In some implementations, this document provides inclusion formula (I): (RL) n -P is an APP RNAi reagent, wherein R is a double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand, wherein the sense strand comprises SEQ ID NO: 35 and the antisense strand comprises SEQ ID NO: 36 or 214; wherein P is a protein comprising a monovalent human TfR binding domain; wherein L is a linker, or optionally absent, wherein the human TfR binding domain comprises two heavy chains HC1 and HC2 and a light chain LC1, wherein HC1 comprises SEQ ID NO: 16, LC1 comprises SEQ ID NO: 10, HC2 comprises SEQ ID NO: 17, and wherein n is 1. In some embodiments, L is a linker from Table 4 (e.g., the SMCC linker from Table 4).

[0027] In some implementations, this document provides inclusion formula (I): (RL) n -P is an APP RNAi reagent, wherein R is a double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand, wherein the sense strand comprises SEQ ID NO: 172 and the antisense strand comprises SEQ ID NO: 173 or 217; wherein P is a protein comprising a monovalent human TfR binding domain; wherein L is a linker, or optionally absent, and wherein n is an integer from 1 to 3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, L is a linker from Table 4 (e.g., the SMCC linker from Table 4).

[0028] In some implementations, this document provides inclusion formula (I): (RL) n-P is an APP RNAi reagent, wherein R is a double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand, wherein the sense strand comprises SEQ ID NO: 172 and the antisense strand comprises SEQ ID NO: 173 or 217; wherein P is a protein comprising a monovalent human TfR binding domain; wherein L is a linker, or optionally absent, wherein the human TfR binding domain comprises two heavy chains HC1 and HC2 and a light chain LC1, wherein HC1 comprises SEQ ID NO: 14, LC1 comprises SEQ ID NO: 10, HC2 comprises SEQ ID NO: 15, and wherein n is 1 or 2. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, L is a linker from Table 4 (e.g., the SMCC linker from Table 4).

[0029] In some implementations, this document provides inclusion formula (I): (RL) n -P is an APP RNAi reagent, wherein R is a double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand, wherein the sense strand comprises SEQ ID NO: 172 and the antisense strand comprises SEQ ID NO: 173 or 217; wherein P is a protein comprising a monovalent human TfR binding domain; wherein L is a linker, or optionally absent, wherein the human TfR binding domain comprises two heavy chains HC1 and HC2 and a light chain LC1, wherein HC1 comprises SEQ ID NO: 16, LC1 comprises SEQ ID NO: 10, HC2 comprises SEQ ID NO: 17, and wherein n is 1. In some embodiments, L is a linker from Table 4 (e.g., the SMCC linker from Table 4).

[0030] In another aspect, this article provides methods for treating APP-related neurological disorders in patients with this need, and such methods include administering an effective amount of the APP RNAi reagent or pharmaceutical composition described herein to the patient. In some embodiments, the APP-related neurological disorder is selected from Alzheimer's disease, Down syndrome, or cerebral amyloid angiopathy. The APP RNAi reagent or pharmaceutical composition containing the APP RNAi reagent can be administered to the patient intravenously or subcutaneously.

[0031] In another aspect, this article provides APP RNAi reagents or pharmaceutical compositions comprising APP RNAi reagents for use in therapies. This article also provides APP RNAi reagents or pharmaceutical compositions comprising APP RNAi reagents for the treatment of APP-related neurological disorders, such as Alzheimer's disease, Down syndrome, or cerebral amyloid angiopathy. This article further provides the use of APP RNAi reagents in the manufacture of pharmaceutical preparations for the treatment of APP-related neurological disorders, such as Alzheimer's disease, Down syndrome, or cerebral amyloid angiopathy. Brief description of the attached diagram Figure 1A An exemplary analytical anion exchange (aAEX) chromatogram is shown, illustrating the DAR profile of the TBP5-dsRNA conjugate number 48 before purification. Figure 1B An exemplary aAEX chromatogram showing the DAR profile of the purified TBP5-dsRNA conjugate number 48 is displayed. Figure 1C An exemplary analytical anion exchange (aAEX) chromatogram is shown, illustrating the DAR profile of the TBP4-dsRNA conjugate number 48 before purification. Figure 1D An exemplary aAEX chromatogram showing the DAR profile of the purified TBP4-dsRNA conjugate number 48 is displayed. Figure 1E An exemplary analytical anion exchange (aAEX) chromatogram is shown, illustrating the DAR profile of the TBP5-dsRNA conjugate number 109 before purification. Figure 1F An exemplary aAEX chromatogram showing the DAR profile of the purified TBP5-dsRNA conjugate number 109 is displayed.

[0033] Figure 2A and 2B The in vitro efficacy of two APP RNAi reagents (TBP4-dsRNA No. 48 in 2A and TBP4-dsRNA No. 50 in 2B) for knocking down the human APP gene (mRNA) in EFO-21 cells was demonstrated. Figure 2C and 2D The in vitro efficacy of two APP RNAi reagents (mTBP1-dsRNA No. 48 in 2C and mTBP1-dsRNA No. 50 in 2D) for knocking down the mouse APP gene (mRNA) in mouse cortical neurons is demonstrated. Figure 2C The dose-response curves yielded the following IC50 fits: cholesterol-conjugated APP dsRNA48 (IC50 = 0.749 nm); IsoAb-dsRNA48-DAR2 (IC50 = 28.3 nM); mTBP1-dsRNA48-DAR2 (IC50 = 0.558 nM). Figure 2DThe dose-response curves yielded the following IC50 fits: cholesterol-conjugated APP dsRNA50 (IC50 = 0.270 nM); IsoAb-dsRNA50-DAR2 (IC50 = 36.6 nM); mTBP1-dsRNA50-DAR2 (IC50 = 0.430 nM).

[0034] Figure 3A and 3B The in vivo efficacy of the two APP RNAi agents was shown after 7 days following a single ICV dose of 30 μg in wild-type mice. The two formulations have different 2' fluorine modification patterns; see Table 7a for dsRNA number 48 and dsRNA number 63. Figure 3A It showed APP gene (mRNA) knockdown, and Figure 3B The knockdown of Aβ (measured for both Aβ(1–40) and Aβ(1–42)) proteins in disease-associated cortical and hippocampal regions was demonstrated using two specified RNAi reagents.

[0035] Figure 4A and 4B The in vivo efficacy of two TfR-binding protein APP siRNA conjugates, TBP4-dsRNA No. 48 (DAR2) or TBP5-sdRNA No. 48 (DAR1), in the brain was demonstrated 28 days after a single (IV) dose of 10 mg / kg (effective dsRNA concentration) in hTfR transgenic mice. Figure 4A The results showed a decrease in APP mRNA in either TBP4-dsRNA number 48 (DAR2) or TBP5-sdRNA number 48 (DAR1) using the APP RNAi reagent. Figure 4B The exposure of the antisense strand of the above-mentioned APP RNAi reagent in the mouse brain was shown.

[0036] Figures 5A-5D The in vivo efficacy of two TfR-binding protein APP siRNA conjugates, TBP4-dsRNA No. 48 (DAR2) or TBP5-sdRNA No. 48 (DAR1), in the brain was demonstrated 28 days after a single (IV) dose of 10 mg / kg (effective dsRNA concentration) in cynomolgus monkeys. Figure 5A The study showed that APP mRNA levels in the brains of cynomolgus monkeys decreased after a single intravenous (IV) dose of the APP RNAi reagent. Figure 5B The study showed a decrease in Aβ protein (both Aβ(1-40) and Aβ(1-42)) after a single intravenous (IV) dose of the APP RNAi reagent. Figure 5CThe study showed the exposure of the antisense strand of the APP RNAi reagent in both the central nervous system (prefrontal cortex, hippocampus) and peripheral (spleen, kidney, liver, heart) tissues of cynomolgus monkeys 28 days after dose. Figure 5D The pharmacokinetic exposure profile of the specified reagent in plasma is shown, along with the concentration (nM) of the antibody-conjugated antisense chain.

[0037] Figures 6A-6D The durability of efficacy of the TfR-binding protein APP siRNA conjugate TBP5-sdRNA number 48 (DAR1) was confirmed at 29, 92, or 181 days after a single (IV) dose of 10 mg / kg (effective dsRNA concentration) in cynomolgus monkeys. Figure 6A and 6C The decrease in APP mRNA in cynomolgus brain following a single intravenous (IV) dose of the APP RNAi reagent in the prefrontal cortex (6A) and hippocampus (6C) is shown (mean ± SEM, n=3). Figure 6B and 6D The decrease in Aβ (measured for both Aβ(1–40) and Aβ(1–42) protein levels after a single intravenous (IV) dose of the APP RNAi reagent in the prefrontal cortex (6B) and hippocampus (6D) is shown (mean ± SEM, n=3).

[0038] Figure 7A and 7B The in vivo efficacy of the APP RNAi reagent, TBP5-sdRNA number 48 (DAR1), following a single intravenous (IV) or subcutaneous (SC) dose, is demonstrated in transgenic hTfR mice. The reagent was administered at 3, 1, or 0.3 mg / kg (effective dsRNA concentration). Figure 7A It was shown that APP mRNA levels decreased in the prefrontal cortex 28 days after the dose; and Figure 7B The study showed that APP mRNA levels in the hippocampus decreased 28 days after the dose was administered.

[0039] Figure 8 The in vivo efficacy of the APP RNAi reagents following a single intravenous (IV) dose of either TBP5-dsRNA number 48 (DAR1) or TBP5-dsRNA number 109 (DAR1) in transgenic hTfR mice is demonstrated. TBP5-dsRNA number 109 (DAR1) has a reverse debasement cap at the 3' end of the antisense strand. The reagents were administered IV to transgenic hTfR mice at a dose of 1 mg / kg (effective dsRNA concentration). Figure 8The study showed reduced APP mRNA levels in the hippocampus, prefrontal cortex, and brainstem at 29 or 84 days post-dose.

[0040] Figure 9A and 9B The potency and persistence of the TfR-binding protein APP siRNA conjugate, TBP5-dsRNA number 109 (DAR1), with a reverse debase cap at the 3' end of the antisense strand, were confirmed in cynomolgus monkeys after a single (IV) dose of 10 mg / kg (effective dsRNA concentration). Figure 9A The study showed a decrease in APP mRNA in disease-related cortical (prefrontal, motor, parietal, and temporal) regions at 29 and 85 days post-dose (mean, n=3–4). Figure 9B The study showed a decrease in Aβ protein (measured both Aβ(1–40) and Aβ(1–42)) in the disease-related cortical (prefrontal, motor, parietal, and temporal lobes) and hippocampal regions at 29 and 85 days post-dose (mean, n=3–4). Invention Details This article provides APP RNAi reagents that can access the central nervous system (CNS) and reduce APP mRNA expression, as well as compositions containing APP RNAi reagents. This article also provides methods for using APP RNAi reagents or compositions containing APP RNAi reagents to reduce APP expression and / or treat APP-related neurological disorders.

[0042] In one respect, this paper provides inclusion equation (I): (RL) n -P is an APP RNAi reagent, wherein R is a double-stranded RNA (dsRNA) containing a sense strand and an antisense strand, wherein the antisense strand is complementary to the APP mRNA; wherein P is a protein containing a monovalent human TfR-binding domain (“human TfR-binding protein”); wherein L is a linker, or optionally absent, and wherein n is an integer from 1 to 3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3.

[0043] In some implementations, this document provides inclusion formula (I): (RL) n-P is an APP RNAi reagent, wherein R is a double-stranded RNA (dsRNA) containing a sense strand and an antisense strand, and said antisense strand is complementary to APP mRNA; wherein P is a protein containing a monovalent human TfR binding domain; wherein L is a linker, or optionally absent, wherein said human TfR binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein said VH comprises heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and said VL comprises light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 3, LCDR1 comprises SEQ ID NO: 4, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 6; and wherein n is an integer from 1 to 3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some implementations, L is the connector in Table 4 (e.g., the SMCC connector in Table 4).

[0044] In some implementations, this document provides inclusion formula (I): (RL) n -P is an APP RNAi reagent, wherein R is a double-stranded RNA (dsRNA) containing a sense strand and an antisense strand, and said antisense strand is complementary to APP mRNA; wherein P is a protein containing a monovalent human TfR binding domain, and P is selected from TBP1, TBP2, TBP3, TBP4, or TBP5 in Table 1b; wherein L is a linker, or optionally absent, and wherein n is 1 or 3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, L is a linker in Table 4 (e.g., the SMCC linker in Table 4).

[0045] In some implementations, this document provides inclusion formula (I): (RL) n -P is an APP RNAi reagent, wherein R is a double-stranded RNA (dsRNA) containing sense and antisense strands, and the dsRNA is any dsRNA in Tables 5a, 5b, 7a, or 7b (e.g., dsRNA number 1); wherein P is a protein containing a monovalent human TfR binding domain; wherein L is a linker, or optionally absent; and wherein n is an integer from 1 to 3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, L is a linker in Table 4 (e.g., the SMCC linker in Table 4).

[0046] In some implementations, this document provides inclusion formula (I): (RL) n -P is an APP RNAi reagent, wherein R is a double-stranded RNA (dsRNA) containing sense and antisense strands, and the dsRNA is any dsRNA in Tables 5a, 5b, 7a, or 7b (e.g., dsRNA number 1); wherein P is a protein containing a monovalent human TfR binding domain, and P is selected from TBP1, TBP2, TBP3, TBP4, or TBP5 in Table 1b; wherein L is a linker, or optionally absent, and wherein n is an integer from 1 to 3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, L is a linker in Table 4 (e.g., the SMCC linker in Table 4).

[0047] In another aspect, this document provides APP RNAi reagents comprising double-stranded RNA (dsRNA) containing a sense strand and an antisense strand, wherein the sense and antisense strand sequences are selected from Tables 5a, 5b, 7a, and 7b. In some embodiments, the APP RNAi reagent comprises any dsRNA from Tables 5a, 5b, 7a, and 7b.

[0048] Human TfR-binding protein The APP RNAi reagent described herein comprises a protein containing a monovalent human TfR binding domain (“human TfR binding protein”). The human TfR binding protein of the APP RNAi reagent can bind to TfR on the BBB and transport dsRNA into the CNS.

[0049] Tables 1a and 1b provide exemplary sequences of human TfR binding domains and proteins. In some embodiments, the monovalent human TfR binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3. In some embodiments, HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 3, LCDR1 comprises SEQ ID NO: 4, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 6. In some embodiments, VH comprises SEQ ID NO: 7, and VL comprises SEQ ID NO: 8. In some embodiments, VH comprises a sequence having at least 95% sequence identity with SEQ ID NO: 7, and VL comprises a sequence having at least 95% sequence identity with SEQ ID NO: 8.

[0050] Table 1a. Exemplary sequences of human TfR binding domains and proteins .

[0051] Table 1b. Exemplary sequences of human TfR-binding proteins .

[0052] In some embodiments, the monovalent human TfR binding domain is an antibody fragment, such as Fab, scFv, Fv, or scFab (single-chain Fab). In some embodiments, the monovalent human TfR binding domain is Fab. In some embodiments, the human TfR binding domain further comprises a heavy chain constant region and / or a light chain constant region.

[0053] In some implementations, the human TfR-binding protein further includes a half-life extension, such as an immunoglobulin Fc region or a VHH that binds to human serum albumin (HSA).

[0054] In some embodiments, the human TfR-binding protein further comprises an immunoglobulin Fc region, such as a modified human IgG4 Fc region or a modified human IgG1 Fc region. In some embodiments, the human TfR-binding protein further comprises a modified human IgG4 Fc region containing proline at residue 228 and alanine at residues 234 and 235 (all residues are numbered according to EU index numbers, also referred to as the hIgG4PAA Fc region). In some embodiments, the human TfR-binding protein further comprises a modified human IgG1 Fc region containing alanine at residues 234, 235, and 329, serine at position 265, and aspartic acid at position 436 (all residues are numbered according to EU index numbers, also referred to as the hIgG1 effector null or hIgG1EN Fc region).

[0055] In some embodiments, the human TfR-binding protein further comprises a VHH that binds to human HSA. In some embodiments, the VHH also binds to mouse, rat, and / or cynomolgus monkey albumin. Exemplary VHHs that bind to human HSA are shown in Table 2. In some embodiments, such VHHs comprise CDR1 comprising SEQ ID NO: 20, CDR2 comprising SEQ ID NO: 21, and CDR3 comprising SEQ ID NO: 22. In some embodiments, such VHHs comprise SEQ ID NO: 23. In some embodiments, the VHH is linked to the TfR-binding domain via a peptide linker such as (GGGGQ)4 (SEQ ID NO: 24). In some embodiments, the VHH is linked to the C-terminus of the TfR-binding domain.

[0056] Table 2. Exemplary sequences of VHH binding to human serum albumin (HSA) .

[0057] In some embodiments, the human TfR-binding protein is a heterodimeric antibody comprising a first arm containing a monovalent human TfR-binding domain and a second arm that is an empty arm, such as an arm that does not bind to any known human target (e.g., an allotype arm). Heterodimeric antibodies, such as heteromonoclonal antibodies, orthothomabs, or duobody, have been described in WO2014150973, WO2016118742, WO2018118616, and WO2011131746. In some embodiments, the first arm contains any monovalent human TfR-binding domain described herein. In some embodiments, the second arm is an empty arm that does not bind to any known human target (e.g., an allotype arm) and contains the sequences in Table 1a. In some embodiments, the second arm comprises a heavy chain (HC) and a light chain (LC), wherein the HC contains SEQ ID NO: 18 and the LC contains SEQ ID NO: 19.

[0058] In some embodiments, the human TfR-binding protein contains a heterodimer mutation. In some embodiments, the human TfR-binding protein includes an Fc region modified with a first Fc CH3 domain and a second Fc CH3 domain, wherein the first Fc CH3 domain comprises serine at residue 349, methionine at residue 366, tyrosine at residue 370, and valine at residue 409, and the second Fc CH3 domain comprises glycine at residue 356, aspartic acid at residue 357, glutamine at residue 364, and alanine at residue 407 (all residues are numbered according to EU index numbers). In some embodiments, the human TfR-binding protein includes an Fc region modified with a first Fc CH3 domain and a second Fc CH3 domain, wherein the first Fc CH3 domain comprises leucine at residue 405, and the second Fc CH3 domain comprises arginine at residue 409 (all residues are numbered according to EU index numbers).

[0059] In some embodiments, the human TfR-binding protein comprises one or more native cysteine ​​residues that can be used for conjugation. For example, in some embodiments, the human TfR-binding protein comprises a native cysteine ​​residue at position 220 of the light chain and / or a native cysteine ​​residue at position 226 of the heavy chain that can be used for conjugation (all residues are numbered according to EU index).

[0060] In some embodiments, the human TfR-binding protein comprises a modified cysteine ​​residue for conjugation. Methods of including modified cysteine ​​residues as a means of conjugation are described in WO 2018 / 232088. In some embodiments, the human TfR-binding protein comprises a heavy chain containing one or more cysteine ​​residues at the following residues: 124, 157, 162, 262, 373, 375, 378, 397, 415 (all residues are according to EU index numbers). In some embodiments, the human TfR-binding protein comprises a light chain (e.g., a κ light chain) containing one or more cysteine ​​residues at the following residues: 156, 171, 191, 193, 202, 208 (all residues are according to EU index numbers). In some embodiments, the human TfR-binding protein comprises a heavy chain constant region containing a cysteine ​​residue at residue 124 (according to EU index number). In some embodiments, the human TfR-binding protein includes a light chain constant region containing a cysteine ​​residue at residue 156 (according to the EU index number). In some embodiments, the human TfR-binding protein includes an immunoglobulin Fc region containing a cysteine ​​residue at residue 378 (according to the EU index number).

[0061] In some implementations, the human TfR binding protein is any one of the human TfR binding proteins in Table 1b, such as TBP1, TBP2, TBP3, TBP4, and TBP5.

[0062] In some embodiments, the human TfR-binding protein has a Fab form, such as TBP1. In some embodiments, the human TfR-binding protein comprises an HC and an LC, wherein the HC comprises SEQ ID NO: 9 and the LC comprises SEQ ID NO: 10.

[0063] In some embodiments, the human TfR binding protein has a Fab-VHH form, such as TBP2. In some embodiments, the human TfR binding protein comprises an HC and an LC, wherein the HC comprises SEQ ID NO: 11 and the LC comprises SEQ ID NO: 12 or 10.

[0064] In some embodiments, the human TfR-binding protein is in the form of a heterodimeric antibody, such as TBP3. In some embodiments, the human TfR-binding protein comprises two heavy chains HC1 and HC2 and two light chains LC1 and LC2, wherein HC1 comprises SEQ ID NO: 13, LC1 comprises SEQ ID NO: 10, HC2 comprises SEQ ID NO: 18, and LC2 comprises SEQ ID NO: 19.

[0065] In some embodiments, the human TfR-binding protein is in the form of a single-arm heterologous monoclonal antibody, such as TBP4 or TBP5. In some embodiments, the human TfR-binding protein comprises two heavy chains HC1 and HC2 and one light chain LC1, wherein HC1 comprises SEQ ID NO: 14, LC1 comprises SEQ ID NO: 10, and HC2 comprises SEQ ID NO: 15. In some embodiments, the human TfR-binding protein provided herein comprises two heavy chains HC1 and HC2 and one light chain LC1, wherein HC1 comprises SEQ ID NO: 16, LC1 comprises SEQ ID NO: 10, and HC2 comprises SEQ ID NO: 17.

[0066] The human TfR-binding protein described herein can be recombinantly generated in host cells, for example, using an expression vector. For instance, the expression vector may include a sequence encoding one or more signal peptides that promote the secretion of the polypeptide from the host cell. Expression vectors containing a polynucleotide of interest (e.g., a polynucleotide encoding the heavy or light chain of a TfR-binding protein) can be transferred into host cells using well-known methods. Additionally, the expression vector may contain one or more selection markers, such as tetracycline, neomycin, and dihydrofolate reductase, to aid in the detection of host cells transformed with the desired polynucleotide sequence.

[0067] Host cells include cells stably or transiently transfected, transformed, transduced, or infected with one or more expression vectors that express all or part of the TfR-binding proteins described herein. According to some embodiments, host cells can be stably or transiently transfected, transformed, transduced, or infected with expression vectors expressing HC peptides and LC peptides of the TfR-binding proteins described herein. In some embodiments, host cells can be stably or transiently transfected, transformed, transduced, or infected with expression vectors expressing both HC and LC peptides of the TfR-binding proteins described herein. TfR-binding proteins can be produced in mammalian cells such as CHO, NSO, HEK293, or COS cells according to techniques well known in the art.

[0068] The culture medium into which TfR-binding proteins have been secreted can be purified using conventional techniques, such as a hybrid approach combining ion exchange and hydrophobic interaction chromatography. For example, the culture medium can be applied to and eluted from a protein A or G column using conventional methods; a hybrid approach combining ion exchange and hydrophobic interaction chromatography can also be used. Soluble aggregates and polymers can be effectively removed using common techniques, including size exclusion, hydrophobic interaction, ion exchange, or hydroxyapatite chromatography. A variety of protein purification methods can be employed, and such methods are known in the art and are described, for example, in Deutscher, Methods in Enzymology 182: 83-89 (1990) and Scopes, Protein Purification: Principles and Practice, 3rd Edition, Springer, NY (1994).

[0069] Mouse TfR binding protein Some of the APP RNAi reagents used in the examples below contain proteins that include a monovalent mouse TfR-binding domain (“mouse TfR-binding protein” or mTBP). Exemplary sequences of the mouse TfR-binding protein are provided in Table 3. Such APP RNAi reagents containing the mouse TfR-binding protein can serve as alternative molecules to APP RNAi reagents containing the human TfR-binding protein in mouse models.

[0070] Table 3. Exemplary sequences of mouse TfR-binding protein (mTBP1) 。

[0071] Linker In some embodiments, the APP RNAi reagents described herein include a linker that links the human TfR-binding protein to the dsRNA. In some embodiments, the linker is a Mal-Tet-TCO linker, an SMCC linker, or a GDM linker (the structures of these linkers are shown in Table 4). In some embodiments, the linker is an SMCC linker.

[0072] Table 4. Exemplary linker structures 。

[0073] dsRNA The APP RNAi reagents described herein include double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand, and wherein the antisense strand is complementary to APP mRNA. After the antisense strand of the dsRNA is incorporated into the RNA-induced silencing complex (RISC), RISC can bind and degrade the target APP mRNA.

[0074] In some embodiments, the sense and antisense strands of the dsRNA are each 15-30 nucleotides in length, such as 20-25 nucleotides in length. In some embodiments, the dsRNA has a sense strand of 21 nucleotides and an antisense strand of 23 nucleotides. In some embodiments, the sense and antisense strands of the dsRNA can have overhangs at the 5'-end or 3'-end (i.e., 5'-overhang or 3'-overhang). For example, the sense and antisense strands can have a 5'- or 3'-overhang of 1 to 5 nucleotides or 1 to 3 nucleotides. In some embodiments, the antisense strand comprises a 3'-overhang of two nucleotides.

[0075] Exemplary unmodified sense and antisense strand sequences of dsRNA targeting human APP mRNA are provided in Tables 5a and 5b.

[0076] Table 5a. Unmodified nucleic acid sequences of dsRNA of the 3' UTR targeting human APP mRNA .

[0077] Table 5b. Unmodified nucleic acid sequences of dsRNAs encoding human APP mRNA. .

[0078] In some implementations, the sense and antisense strands of the dsRNA comprise a pair of nucleic acid sequences selected from the following: (a) The sense chain contains SEQ ID NO: 35, and the antisense chain contains SEQ ID NO: 36; (b) The sense chain contains SEQ ID NO: 37, and the antisense chain contains SEQ ID NO: 38; (c) The sense chain contains SEQ ID NO: 39, and the antisense chain contains SEQ ID NO: 40; (d) The sense chain contains SEQ ID NO: 41, and the antisense chain contains SEQ ID NO: 42; (e) The sense chain contains SEQ ID NO: 43, and the antisense chain contains SEQ ID NO: 44; (f) The sense chain contains SEQ ID NO: 45, and the antisense chain contains SEQ ID NO: 46; (g) The sense chain contains SEQ ID NO: 47, and the antisense chain contains SEQ ID NO: 48; (h) The sense chain contains SEQ ID NO: 49, and the antisense chain contains SEQ ID NO: 50; (i) The sense chain contains SEQ ID NO: 51, and the antisense chain contains SEQ ID NO: 52; (j) The sense chain contains SEQ ID NO: 53, and the antisense chain contains SEQ ID NO: 54; (k) The sense chain contains SEQ ID NO: 55, and the antisense chain contains SEQ ID NO: 56; (l) The sense chain contains SEQ ID NO: 57, and the antisense chain contains SEQ ID NO: 58; (m) The sense chain contains SEQ ID NO: 59, and the antisense chain contains SEQ ID NO: 60; (n) The sense chain contains SEQ ID NO: 61, and the antisense chain contains SEQ ID NO: 62; (o) The sense chain contains SEQ ID NO: 63, and the antisense chain contains SEQ ID NO: 64; (p) The sense chain contains SEQ ID NO: 65, and the antisense chain contains SEQ ID NO: 66; (q) The sense chain contains SEQ ID NO: 67, and the antisense chain contains SEQ ID NO: 68; (r) The sense chain contains SEQ ID NO: 69, and the antisense chain contains SEQ ID NO: 70; (s) The sense chain contains SEQ ID NO: 71, and the antisense chain contains SEQ ID NO: 72; (t) The sense chain contains SEQ ID NO: 73, and the antisense chain contains SEQ ID NO: 74; (u) The sense chain contains SEQ ID NO: 75, and the antisense chain contains SEQ ID NO: 76; (v) The sense chain contains SEQ ID NO: 77, and the antisense chain contains SEQ ID NO: 78; (w) The sense chain contains SEQ ID NO: 79, and the antisense chain contains SEQ ID NO: 80; (x) The sense chain contains SEQ ID NO: 81, and the antisense chain contains SEQ ID NO: 82; (y) The sense chain contains SEQ ID NO: 83, and the antisense chain contains SEQ ID NO: 84; (z) The sense chain contains SEQ ID NO: 85, and the antisense chain contains SEQ ID NO: 86; (aa) The sense chain contains SEQ ID NO: 87, and the antisense chain contains SEQ ID NO: 88; (bb) The sense chain contains SEQ ID NO: 89, and the antisense chain contains SEQ ID NO: 90; (cc) The sense chain contains SEQ ID NO: 91, and the antisense chain contains SEQ ID NO: 92; (dd) The sense chain contains SEQ ID NO: 93, and the antisense chain contains SEQ ID NO: 94; (ee) The sense chain contains SEQ ID NO: 95, and the antisense chain contains SEQ ID NO: 96; (ff) The sense chain contains SEQ ID NO: 97, and the antisense chain contains SEQ ID NO: 98; (gg) The sense chain contains SEQ ID NO: 99, and the antisense chain contains SEQ ID NO: 100; (hh) The sense chain contains SEQ ID NO: 184, and the antisense chain contains SEQ ID NO: 36; (ii) The sense chain contains SEQ ID NO: 188, and the antisense chain contains SEQ ID NO: 38; and (jj) The sense chain contains SEQ ID NO: 35, and the antisense chain contains SEQ ID NO: 214; Optionally, one or more nucleotides of the sense and antisense strands are independently modified nucleotides, and the bonding between one or more nucleotides of the sense and antisense strands is a modified nucleotide bonding. In some embodiments, the sense strand comprises SEQ ID NO: 35, and the antisense strand comprises SEQ ID NO: 36. In some embodiments, the sense strand comprises SEQ ID NO: 35, and the antisense strand comprises SEQ ID NO: 214. In some embodiments, the sense strand comprises SEQ ID NO: 37, and the antisense strand comprises SEQ ID NO: 38.

[0079] dsRNA can include modifications. Modifications can be made to one or more nucleotides of the sense and / or antisense strand, or to internucleotide bonds, which are bonds between two nucleotides in the sense or antisense strand. For example, some 2'-modifications of ribose or deoxyribose can increase the stability and half-life of RNA or DNA. Such 2'-modifications can be 2'-fluorine, 2'-O-methyl (i.e., 2'-methoxy), or 2'-O-alkyl.

[0080] In some embodiments, one or more nucleotides of the sense strand and / or antisense strand are independently modified nucleotides, meaning that the sense strand and antisense strand can have different modified nucleotides. In some embodiments, each nucleotide of the sense strand is a modified nucleotide. In some embodiments, at least one nucleotide of the sense strand is an unmodified RNA nucleotide. In some embodiments, each nucleotide of the antisense strand is a modified nucleotide. In some embodiments, the modified nucleotide is a 2'-fluorinated nucleotide, a 2'-O-methylated nucleotide, a 2'-deoxynucleotide (DNA), or a 2'-O-alkylated nucleotide. In some embodiments, each nucleotide of the sense strand and antisense strand is an independently modified nucleotide, such as a 2'-fluorinated nucleotide, a 2'-O-methylated nucleotide, a 2'-deoxynucleotide (DNA), or a 2'-O-alkylated nucleotide. In some embodiments, at least one nucleotide of the sense strand is a 2'-deoxynucleotide (DNA).

[0081] In some embodiments, the sense strand has, for example, four 2'-fluorinated nucleotides at positions 7, 9, 10, and 11 from the 5' end of the sense strand. In some embodiments, at least one nucleotide of the sense strand is an unmodified RNA nucleotide. In some embodiments, at least one nucleotide of the sense strand is a 2' deoxyribonucleotide (DNA). In some embodiments, other nucleotides of the sense strand are 2'-O-methyl modified nucleotides.

[0082] In some embodiments, the antisense strand has, for example, four 2'-fluorinated nucleotides at positions 2, 6, 14, and 16 from the 5' end of the antisense strand. In some embodiments, the other nucleotides of the antisense strand are 2'-O-methyl nucleotides.

[0083] In some embodiments, the sense strand has, for example, three 2'-fluorinated nucleotides at positions 9, 10, and 11 from the 5' end of the sense strand. In some embodiments, at least one nucleotide of the sense strand is an unmodified RNA nucleotide. In some embodiments, at least one nucleotide of the sense strand is a 2' deoxyribonucleotide (DNA). In some embodiments, other nucleotides of the sense strand are 2'-O-methyl modified nucleotides.

[0084] In some embodiments, the antisense strand has, for example, five 2'-fluorinated nucleotides at positions 2, 5, 7, 14, and 16 from the 5' end of the antisense strand. In some embodiments, the antisense strand has, for example, five 2'-fluorinated nucleotides at positions 2, 5, 8, 14, and 16 from the 5' end of the antisense strand. In some embodiments, the other nucleotides of the antisense strand are 2'-O-methyl modified nucleotides.

[0085] In some implementations, the 5' end of the antisense chain has a phosphate analog, such as 5'-vinylphosphonate (5'-VP).

[0086] In some embodiments, the sense or antisense strand includes a debasement moiety or a reverse debasement moiety, such as those shown in Table 6. In some embodiments, the reverse debasement moiety or debasement moiety increases the stability of the sense or antisense strand. In some embodiments, the sense strand includes a reverse debasement moiety. In some embodiments, the antisense strand includes a reverse debasement moiety.

[0087] Table 6. Debasement or Reverse Debasement (iAb) Section “5’” and “3’” indicate the 5’ to 3’ direction of the sequence.

[0088] In some embodiments, the sense strand and antisense strand have one or more modified nucleotide bonds. In some embodiments, the modified nucleotide bonds are phosphate thioester bonds. In some embodiments, the sense strand has four or five phosphate thioester bonds. In some embodiments, the antisense strand has four or five phosphate thioester bonds. In some embodiments, both the sense strand and antisense strand have four or five phosphate thioester bonds. In some embodiments, the sense strand has four phosphate thioester bonds, and the antisense strand has five phosphate thioester bonds.

[0089] Tables 7a and 7b provide exemplary sense and antisense strand sequences of modified dsRNA targeting human APP mRNA.

[0090] In some embodiments, the dsRNA contains a sense strand that includes a sequence that differs from the sense strand sequences in Table 7a or 7b by one, two, or three times. In some embodiments, the dsRNA contains an antisense strand that includes a sequence that differs from the antisense strand sequences in Table 7a or 7b by one, two, or three times.

[0091] Table 7a: Nucleic acid sequences modified by dsRNA targeting the 3'UTR of human APP mRNA Abbreviations - “m” indicates 2'-OMe; “f” indicates 2'-fluorine; “*” indicates thiophosphate bond; “VP” indicates 5'-vinylphosphonate; “iAb” indicates the reverse debasing moiety in Table 6; “n” indicates the debasing moiety; “d” indicates 2'-deoxygenation; “S” indicates sense chain; “AS” indicates antisense chain; unless otherwise specified, the 5' position of AS may include 5'-phosphate or 5'-vinylphosphonate.

[0092] Table 7b: Nucleic acid sequences modified with dsRNA that target human APP mRNA coding sequence Abbreviations - “m” indicates 2'-OMe; “f” indicates 2'-fluorine; “*” indicates thiophosphate bond; “VP” indicates 5'-vinylphosphonate; “iAb” indicates the reverse debasement portion in Table 6; “S” indicates sense chain; “AS” indicates antisense chain; unless otherwise stated, the 5' position of AS may include 5'-phosphate or 5'-vinylphosphonate.

[0093] In some implementations, the sense and antisense strands of the dsRNA comprise a pair of nucleic acid sequences selected from the following: (a) The sense chain contains SEQ ID NO: 101, and the antisense chain contains SEQ ID NO: 102, 173, 176, 177, 178, 179, 180, 182 or 185; (b) The sense chain contains SEQ ID NO: 103, and the antisense chain contains SEQ ID NO: 104, 175, 189, 190, 191, 192, 194, 195 or 196; (c) The sense chain contains SEQ ID NO: 105, and the antisense chain contains SEQ ID NO: 106; (d) The sense chain contains SEQ ID NO: 107, and the antisense chain contains SEQ ID NO: 108; (e) The sense chain contains SEQ ID NO: 109, and the antisense chain contains SEQ ID NO: 110; (f) The sense chain contains SEQ ID NO: 111, and the antisense chain contains SEQ ID NO: 112; (g) The sense chain contains SEQ ID NO: 113, and the antisense chain contains SEQ ID NO: 114; (h) The sense chain contains SEQ ID NO: 115, and the antisense chain contains SEQ ID NO: 116; (i) The sense chain contains SEQ ID NO: 117, and the antisense chain contains SEQ ID NO: 118; (j) The sense chain contains SEQ ID NO: 119, and the antisense chain contains SEQ ID NO: 120; (k) The sense chain contains SEQ ID NO: 121, and the antisense chain contains SEQ ID NO: 122; (l) The sense chain contains SEQ ID NO: 123, and the antisense chain contains SEQ ID NO: 124; (m) The sense chain contains SEQ ID NO: 125, and the antisense chain contains SEQ ID NO: 126; (n) The sense chain contains SEQ ID NO: 127, and the antisense chain contains SEQ ID NO: 128; (o) The sense chain contains SEQ ID NO: 129, and the antisense chain contains SEQ ID NO: 130; (p) The sense chain contains SEQ ID NO: 131, and the antisense chain contains SEQ ID NO: 132; (q) The sense chain contains SEQ ID NO: 133, and the antisense chain contains SEQ ID NO: 134; (r) The sense chain contains SEQ ID NO: 135, and the antisense chain contains SEQ ID NO: 136; (s) The sense chain contains SEQ ID NO: 137, and the antisense chain contains SEQ ID NO: 138; (t) The sense chain contains SEQ ID NO: 139, and the antisense chain contains SEQ ID NO: 140; (u) The sense chain contains SEQ ID NO: 141, and the antisense chain contains SEQ ID NO: 142; (v) The sense chain contains SEQ ID NO: 143, and the antisense chain contains SEQ ID NO: 144; (w) The sense chain contains SEQ ID NO: 145, and the antisense chain contains SEQ ID NO: 146; (x) The sense chain contains SEQ ID NO: 147, and the antisense chain contains SEQ ID NO: 148; (y) The sense chain contains SEQ ID NO: 149, and the antisense chain contains SEQ ID NO: 150; (z) The sense chain contains SEQ ID NO: 151, and the antisense chain contains SEQ ID NO: 152; (aa) The sense chain contains SEQ ID NO: 153, and the antisense chain contains SEQ ID NO: 154; (bb) The sense chain contains SEQ ID NO: 155, and the antisense chain contains SEQ ID NO: 156; (cc) The sense chain contains SEQ ID NO: 157, and the antisense chain contains SEQ ID NO: 158; (dd) The sense chain contains SEQ ID NO: 159, and the antisense chain contains SEQ ID NO: 160; (ee) The sense chain contains SEQ ID NO: 161, and the antisense chain contains SEQ ID NO: 162; (ff) The sense chain contains SEQ ID NO: 163, and the antisense chain contains SEQ ID NO: 164; (gg) The sense chain contains SEQ ID NO: 165, and the antisense chain contains SEQ ID NO: 166; (hh) The sense chain contains SEQ ID NO: 172, and the antisense chain contains SEQ ID NO: 173; (ii) The sense chain contains SEQ ID NO: 181, and the antisense chain contains SEQ ID NO: 173; (jj) The sense chain contains SEQ ID NO: 174 or 193, and the antisense chain contains SEQ ID NO: 175; (kk) The sense chain contains SEQ ID NO: 172, and the antisense chain contains SEQ ID NO: 177; (ll) The sense chain contains SEQ ID NO: 181, 183 or 186, and the antisense chain contains SEQ ID NO: 102; (mm) The sense chain contains SEQ ID NO: 187, 193 or 197, and the antisense chain contains SEQ ID NO: 104; (nn) The sense chain contains SEQ ID NO: 198, and the antisense chain contains SEQ ID NO: 199; (oo) The sense chain contains SEQ ID NO: 200, and the antisense chain contains SEQ ID NO: 201; (pp) The sense chain contains SEQ ID NO: 202, and the antisense chain contains SEQ ID NO: 203; (qq) The sense chain contains SEQ ID NO: 204, and the antisense chain contains SEQ ID NO: 205; (rr) The sense chain contains SEQ ID NO: 206, and the antisense chain contains SEQ ID NO: 207; (ss) The sense chain contains SEQ ID NO: 208, and the antisense chain contains SEQ ID NO: 209; (tt) The sense chain contains SEQ ID NO: 210, and the antisense chain contains SEQ ID NO: 118; The (uu) sense chain contains SEQ ID NO: 211, and the antisense chain contains SEQ ID NO: 120; (vv) The sense chain contains SEQ ID NO: 212, and the antisense chain contains SEQ ID NO: 122; (ww) The sense chain contains SEQ ID NO: 213, and the antisense chain contains SEQ ID NO: 124; (xx) The sense chain contains SEQ ID NO: 172, and the antisense chain contains SEQ ID NO: 215; (yy) The sense chain contains SEQ ID NO: 172, and the antisense chain contains SEQ ID NO: 216; (zz) The sense chain contains SEQ ID NO: 172, and the antisense chain contains SEQ ID NO: 217; The (aaa) sense chain contains SEQ ID NO: 172, and the antisense chain contains SEQ ID NO: 218; (bbb) The sense chain contains SEQ ID NO: 219, and the antisense chain contains SEQ ID NO: 220; (ccc) The sense chain contains SEQ ID NO: 221, and the antisense chain contains SEQ ID NO: 222; (ddd) The sense chain contains SEQ ID NO: 223, and the antisense chain contains SEQ ID NO: 224; The (eee) sense chain contains SEQ ID NO: 225, and the antisense chain contains SEQ ID NO: 226; (fff) The sense chain contains SEQ ID NO: 227, and the antisense chain contains SEQ ID NO: 228; The (ggg) sense chain contains SEQ ID NO: 229, and the antisense chain contains SEQ ID NO: 230; (hhh) The sense chain contains SEQ ID NO: 231, and the antisense chain contains SEQ ID NO: 232; (iii) The sense chain contains SEQ ID NO: 233, and the antisense chain contains SEQ ID NO: 234; (jjj) has a sense chain containing SEQ ID NO: 235 and an antisense chain containing SEQ ID NO: 236; (kkk) The sense chain contains SEQ ID NO: 237, and the antisense chain contains SEQ ID NO: 238; (lll) The sense chain contains SEQ ID NO: 239, and the antisense chain contains SEQ ID NO: 240; and (mmm) The sense chain contains SEQ ID NO: 241, and the antisense chain contains SEQ ID NO: 242.

[0094] In some implementations, the sense and antisense strands of the dsRNA have a pair of nucleic acid sequences selected from the following: (a) The sense chain consists of SEQ ID NO: 101, and the antisense chain consists of SEQ ID NO: 102, 173, 176, 177, 178, 179, 180, 182 or 185; (b) The sense chain consists of SEQ ID NO: 103, and the antisense chain consists of SEQ ID NO: 104, 175, 189, 190, 191, 192, 194, 195 or 196; (c) The sense chain consists of SEQ ID NO: 105, and the antisense chain consists of SEQ ID NO: 106; (d) The sense chain consists of SEQ ID NO: 107, and the antisense chain consists of SEQ ID NO: 108; (e) The sense chain consists of SEQ ID NO: 109, and the antisense chain consists of SEQ ID NO: 110; (f) The sense chain consists of SEQ ID NO: 111, and the antisense chain consists of SEQ ID NO: 112; (g) The sense chain consists of SEQ ID NO: 113, and the antisense chain consists of SEQ ID NO: 114; (h) The sense chain consists of SEQ ID NO: 115, and the antisense chain consists of SEQ ID NO: 116; (i) The sense chain consists of SEQ ID NO: 117, and the antisense chain consists of SEQ ID NO: 118; (j) The sense chain consists of SEQ ID NO: 119, and the antisense chain consists of SEQ ID NO: 120; (k) The sense chain consists of SEQ ID NO: 121, and the antisense chain consists of SEQ ID NO: 122; (l) The sense chain consists of SEQ ID NO: 123, and the antisense chain consists of SEQ ID NO: 124; (m) The sense chain consists of SEQ ID NO: 125, and the antisense chain consists of SEQ ID NO: 126; (n) The sense chain consists of SEQ ID NO: 127, and the antisense chain consists of SEQ ID NO: 128; (o) The sense chain consists of SEQ ID NO: 129, and the antisense chain consists of SEQ ID NO: 130; (p) The sense chain consists of SEQ ID NO: 131, and the antisense chain consists of SEQ ID NO: 132; (q) The sense chain consists of SEQ ID NO: 133, and the antisense chain consists of SEQ ID NO: 134; (r) The sense chain consists of SEQ ID NO: 135, and the antisense chain consists of SEQ ID NO: 136; (s) The sense chain consists of SEQ ID NO: 137, and the antisense chain consists of SEQ ID NO: 138; (t) The sense chain consists of SEQ ID NO: 139, and the antisense chain consists of SEQ ID NO: 140; (u) The sense chain consists of SEQ ID NO: 141, and the antisense chain consists of SEQ ID NO: 142; (v) The sense chain consists of SEQ ID NO: 143, and the antisense chain consists of SEQ ID NO: 144; (w) The sense chain consists of SEQ ID NO: 145, and the antisense chain consists of SEQ ID NO: 146; (x) The sense chain consists of SEQ ID NO: 147, and the antisense chain consists of SEQ ID NO: 148; (y) The sense chain consists of SEQ ID NO: 149, and the antisense chain consists of SEQ ID NO: 150; (z) The sense chain consists of SEQ ID NO: 151, and the antisense chain consists of SEQ ID NO: 152; (aa) The sense chain consists of SEQ ID NO: 153, and the antisense chain consists of SEQ ID NO: 154; (bb) The sense chain consists of SEQ ID NO: 155, and the antisense chain consists of SEQ ID NO: 156; (cc) The sense chain consists of SEQ ID NO: 157, and the antisense chain consists of SEQ ID NO: 158; (dd) The sense chain consists of SEQ ID NO: 159, and the antisense chain consists of SEQ ID NO: 160; (ee) The sense chain consists of SEQ ID NO: 161, and the antisense chain consists of SEQ ID NO: 162; (ff) The sense chain consists of SEQ ID NO: 163, and the antisense chain consists of SEQ ID NO: 164; (gg) The sense chain consists of SEQ ID NO: 165, and the antisense chain consists of SEQ ID NO: 166; (hh) The sense chain consists of SEQ ID NO: 172, and the antisense chain consists of SEQ ID NO: 173; (ii) The sense chain consists of SEQ ID NO: 181, and the antisense chain consists of SEQ ID NO: 173; (jj) The sense chain consists of SEQ ID NO: 174 or 193, and the antisense chain consists of SEQ ID NO: 175; (kk) The sense chain consists of SEQ ID NO: 172, and the antisense chain consists of SEQ ID NO: 177; (ll) The sense chain consists of SEQ ID NO: 181, 183 or 186, and the antisense chain consists of SEQ ID NO: 102; (mm) The sense chain consists of SEQ ID NO: 187, 193 or 197, and the antisense chain consists of SEQ ID NO: 104; (nn) The sense chain consists of SEQ ID NO: 198, and the antisense chain consists of SEQ ID NO: 199; (oo) The sense chain consists of SEQ ID NO: 200, and the antisense chain consists of SEQ ID NO: 201; (pp) The sense chain consists of SEQ ID NO: 202, and the antisense chain consists of SEQ ID NO: 203; (qq) The sense chain consists of SEQ ID NO: 204, and the antisense chain consists of SEQ ID NO: 205; (rr) The sense chain consists of SEQ ID NO: 206, and the antisense chain consists of SEQ ID NO: 207; (ss) The sense chain consists of SEQ ID NO: 208, and the antisense chain consists of SEQ ID NO: 209; (tt) The sense chain consists of SEQ ID NO: 210, and the antisense chain consists of SEQ ID NO: 118; The (uu) sense chain consists of SEQ ID NO: 211, and the antisense chain consists of SEQ ID NO: 120; (vv) The sense chain consists of SEQ ID NO: 212, and the antisense chain consists of SEQ ID NO: 122; (ww) The sense chain consists of SEQ ID NO: 213, and the antisense chain consists of SEQ ID NO: 124; (xx) The sense chain consists of SEQ ID NO: 172, and the antisense chain consists of SEQ ID NO: 215; (yy) The sense chain consists of SEQ ID NO: 172, and the antisense chain consists of SEQ ID NO: 216; (zz) The sense chain consists of SEQ ID NO: 172, and the antisense chain consists of SEQ ID NO: 217; The (aaa) sense chain consists of SEQ ID NO: 172, and the antisense chain consists of SEQ ID NO: 218; (bbb) The sense chain consists of SEQ ID NO: 219, and the antisense chain consists of SEQ ID NO: 220; (ccc) The sense chain consists of SEQ ID NO: 221, and the antisense chain consists of SEQ ID NO: 222; (ddd) The sense chain consists of SEQ ID NO: 223, and the antisense chain consists of SEQ ID NO: 224; The (eee) sense chain consists of SEQ ID NO: 225, and the antisense chain consists of SEQ ID NO: 226; (fff) The sense chain consists of SEQ ID NO: 227, and the antisense chain consists of SEQ ID NO: 228; The (ggg) sense chain consists of SEQ ID NO: 229, and the antisense chain consists of SEQ ID NO: 230; (hhh) The sense chain consists of SEQ ID NO: 231, and the antisense chain consists of SEQ ID NO: 232; (iii) The sense chain consists of SEQ ID NO: 233, and the antisense chain consists of SEQ ID NO: 234; The (jjj) sense chain consists of SEQ ID NO: 235, and the antisense chain consists of SEQ ID NO: 236; (kkk) The sense chain consists of SEQ ID NO: 237, and the antisense chain consists of SEQ ID NO: 238; (lll) The sense chain consists of SEQ ID NO: 239, and the antisense chain consists of SEQ ID NO: 240; and (mmm) The sense chain consists of SEQ ID NO: 241, and the antisense chain consists of SEQ ID NO: 242.

[0095] In some embodiments, the sense chain comprises SEQ ID NO: 172, and the antisense chain comprises SEQ ID NO: 217. In some embodiments, the sense chain consists of SEQ ID NO: 172, and the antisense chain consists of SEQ ID NO: 217.

[0096] The sense and antisense strands of dsRNA can be synthesized using any nucleic acid polymerization method known in the art, such as solid-phase synthesis employing phosphoramidite chemistry (e.g., Current Protocols in Nucleic Acid Chemistry, Beaucage, SL et al. (eds.), John Wiley & Sons, Inc., New York, NY, USA), H-phosphonate, phosphate triester chemistry, or enzymatic synthesis. Automated commercial synthesizers can be used, such as the MerMade™ 12 from LGC Biosearch Technologies, or other synthesizers from BioAutomation or Applied Biosystems. Thiophosphate bonds can be introduced using sulfiding agents such as phenylacetyl disulfide or DDTT (((dimethylaminomethylene)amino)-3H-1,2,4-dithiazoline-3-thione). The synthesis of modified or conjugated oligonucleotides using similar techniques and commercially available modified amidites and controlled-pore glass (CPG) products is well known.

[0097] Purification methods are used to remove unwanted impurities from the final oligonucleotide product. Common purification techniques for single-stranded oligonucleotides include reversed-phase ion-pair high-performance liquid chromatography (RP-IP-HPLC), capillary gel electrophoresis (CGE), anion-exchange HPLC (AX-HPLC), and size exclusion chromatography (SEC). After purification, the oligonucleotides can be analyzed by mass spectrometry and quantified spectrophotometrically at a wavelength of 260 nm. The sense and antisense strands can then be annealed to form dsRNA.

[0098] The RNAi reagents described herein can be prepared using various procedures known to those skilled in the art, some of which are illustrated in the preparation and examples below, such as in Examples 1-3. Those skilled in the art will recognize that the specific synthetic steps for each of these pathways can be combined in different ways, or combined with steps from different schemes, to prepare the RNAi reagents. The products of each step can be recovered using conventional methods well known in the art, including extraction, evaporation, precipitation, chromatography, filtration, grinding, and crystallization. The reagents and raw materials are readily available to those skilled in the art.

[0099] In some embodiments, the TfR-binding protein with natural or modified cysteine ​​residues described herein can be first treated with a reducing agent such as DTT, and then re-oxidized with an oxidizing agent such as DHAA. The resulting oxidized TfR-binding protein is then incubated with a linker-functionalized dsRNA, such as a linker-dsRNA, to produce a conjugated RNAi reagent.

[0100] Pharmaceutical Composition In another aspect, this document provides pharmaceutical compositions comprising any of the APP RNAi reagents described herein and pharmaceutically acceptable carriers. Such pharmaceutical compositions may also comprise one or more pharmaceutically acceptable excipients, diluents, or carriers. Pharmaceutical compositions can be prepared using methods well known in the art (e.g., Remington: The Science and Practice of Pharmacy, 23rd edition (2020), A. Loyd et al., Academic Press).

[0101] Treatment methods and therapeutic uses In another aspect, this article provides methods for treating APP-related neurological disorders in patients with this need, and such methods include administering an effective amount of the APP RNAi reagent or pharmaceutical composition described herein to the patient. In some embodiments, the APP-related neurological disorder is selected from Alzheimer's disease, Down syndrome, or cerebral amyloid angiopathy. The APP RNAi reagent or pharmaceutical composition containing the APP RNAi reagent can be administered to the patient intravenously or subcutaneously.

[0102] The APP RNAi reagent dosing regimen can be adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single bolus can be administered, several fractionated doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the urgency of the treatment situation.

[0103] Dosage values ​​may vary depending on the type and severity of the condition to be alleviated. It should be further understood that, for any given subject, the specific dosing regimen should be adjusted over time based on individual needs and the professional judgment of the person administering or supervising the administration of the composition.

[0104] In another aspect, this article provides APP RNAi reagents or pharmaceutical compositions comprising APP RNAi reagents for use in therapies. This article also provides APP RNAi reagents or pharmaceutical compositions comprising APP RNAi reagents for the treatment of APP-related neurological disorders, such as Alzheimer's disease, Down syndrome, or cerebral amyloid angiopathy. This article further provides the use of APP RNAi reagents in the manufacture of pharmaceutical preparations for the treatment of APP-related neurological disorders, such as Alzheimer's disease, Down syndrome, or cerebral amyloid angiopathy.

[0105] definition As used herein, the terms “a,” “an,” “the,” and similar terms used in the context of this disclosure (especially in the context of the claims) should be interpreted to cover both the singular and the plural, unless otherwise stated herein or clearly contradicted by the context.

[0106] As used herein, the term “about” means within a reasonable range of the value, such as plus or minus 10% of the value.

[0107] As used herein, the term "alkyl" refers to a saturated linear or branched monovalent hydrocarbon group containing an indicated number of carbon atoms. For example, "C1-C 20 "Alkyl" refers to a group of atoms having 1-20 carbon atoms arranged in a linear or branched manner.

[0108] As used herein, the term "antibody" refers to a molecule that binds to an antigen. Antibody implementations include monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, heterodimeric antibodies, bispecific or multispecific antibodies, or conjugated antibodies. Antibodies can be of any class (e.g., IgG, IgE, IgM, IgD, IgA) and any subclass (e.g., IgG1, IgG2, IgG3, IgG4).

[0109] Immunoglobulin G (IgG) antibodies consist of four polypeptide chains: two heavy chains (HC) and two light chains (LC) cross-linked via interchain disulfide bonds. The amino-terminal portion of each of the four polypeptide chains contains a variable region of approximately 100-125 or more amino acids primarily responsible for antigen recognition. The carboxyl-terminal portion of each of the four polypeptide chains contains a constant region primarily responsible for effector function. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region. Each light chain consists of a light chain variable region (VL) and a light chain constant region. IgG isotypes can be further subdivided into subtypes (e.g., IgG1, IgG2, IgG3, and IgG4).

[0110] The VH and VL regions can be further subdivided into highly variable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). CDRs are exposed on the protein surface and are crucial regions for antibody-antigen binding specificity. Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In this paper, the three CDRs of the heavy chain are referred to as "HCDR1, HCDR2, and HCDR3," and the three CDRs of the light chain are referred to as "LCDR1, LCDR2, and LCDR3." CDRs contain most of the residues that specifically interact with the antigen. The assignment of amino acid residues to the CDR can be accomplished according to well-known protocols, including those described below: Kabat (Kabat et al., “Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, Md. (1991)), Chothia (Chothia et al., “Canonical structures for the hypervariable regions of immunoglobulins,” Journal of Molecular Biology, 196, 901-917 (1987); Al-Lazikani et al., “Standard conformations for the canonical structures of immunoglobulins,” Journal of Molecular Biology, 273, 927-948 (1997)), North (North et al., “A New Clustering of Antibody CDR Loop Conformations,” Journal of Molecular Biology, 406, 228-256 (2011)), or IMGT. (The international ImMunoGeneTics database is available at www.imgt.org; see Lefranc et al., Nucleic Acids Res. 1999; 27:209-212).

[0111] Embodiments of this disclosure also include antibody fragments or antigen-binding fragments, as used herein, comprising at least a portion of an antibody that retains the ability to specifically interact with an antigen or antigenic epitope, such as Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, scFab, disulfide-linked Fv (sdFv), and Fd fragments.

[0112] As used herein, the term "antigen-binding domain" refers to a portion of an antibody or antibody fragment that binds to an antigen or antigenic epitope. For example, "TfR-binding domain" refers to a portion of an antibody or antibody fragment that binds to a TfR or TfR epitope.

[0113] As used in this article, the term "heterodimeric antibody" refers to an antibody that contains two different antigen-binding domains.

[0114] As used herein, “antisense strand” refers to a single-stranded oligonucleotide that is complementary to a region of the target sequence. Similarly, as used herein, “sense strand” refers to a single-stranded oligonucleotide that is complementary to a region of the antisense strand.

[0115] As used in this article, “APP” (also known as β-amyloid precursor protein or ABPP) refers to the mRNA transcript, protein, or polypeptide of amyloid precursor protein (APP). The nucleotide sequences of human APP mRNA transcript variants and the amino acid sequences of human APP protein isotypes can be found at: a. NM_000484.4 transcript variant 1 → NP_000475.1 APP protein isotype a (longest isotype); b. NM_201413.3 transcript variant 2 → NP_958816.1 APP protein isotype b; c. NM_201414.3 transcript variant 3 → NP_958817.1 APP protein isotype c; d. NM_001136016.3 transcript variant 4 → NP_001129488.1 APP protein isotype d; e. NM_001136129.3 transcript variant 5 → NP_001129601.1 APP protein isotype e; f. NM_001136130.3 transcript variant 6 → NP_001129602.1 APP protein isotype f; g. NM_001136131.3 transcript variant 7 → NP_001129603.1 APP protein isotype g; h. NM_001204301.2 transcript variant 8 → NP_001191230.1 APP protein isotype h; i. NM_001204302.2 transcript variant 9 → NP_001191231.1 APP protein isotype i; j. NM_001204303.2 transcript variant 10 → NP_001191232.1 APP protein isotype j; k. NM_001385253.1 transcript variant 11 → NP_001372182.1 APP protein isotype k.

[0116] The amino acid sequence of human APP protein isotype a (longest isotype) can be found at NP_000475.1: (SEQ ID NO: 167).

[0117] The sequence of human APP mRNA transcript variant 1, which encodes human APP protein isotype a (longest isotype), can be found at NM_000484.4: (SEQ ID NO: 168).

[0118] The nucleic acid sequence of mouse APP mRNA transcript can be found at NM_001198823.1; and the amino acid sequence of mouse APP protein can be found at NP_001185752.1. The nucleic acid sequence of rat APP mRNA transcript can be found at NM_019288.2; and the amino acid sequence of rat APP protein can be found at NP_062161.1. The nucleic acid sequence of monkey APP mRNA transcript can be found at XM_015133068.2; and the amino acid sequence of monkey APP protein can be found at XP_014988554.1.

[0119] As used in this article, the term "APP-related neurological disorders" refers to neurological disorders characterized by extracellular amyloid deposits or plaques.

[0120] Unless otherwise stated, the terms “bind” and “binds” as used herein are intended to mean the ability of a protein or molecule to form a chemical bond or attractive interaction with another protein or molecule, resulting in the proximity of the two proteins or molecules, as determined by common methods known in the art.

[0121] As used herein, “complementary” means a structural relationship between two nucleotides (e.g., on two opposite nucleic acids or on opposite regions of a single nucleic acid strand, such as a hairpin) that allows the two nucleotides to form base pairs with each other. For example, a purine nucleotide of a nucleic acid complementary to a pyrimidine nucleotide of an opposite nucleic acid can be base-paired together by forming hydrogen bonds with each other. Complementary nucleotides can be base-paired in a Watson-Crick manner or in any other manner that allows for the formation of a stable duplex. Similarly, two nucleic acids can have multiple nucleotide regions that are complementary to each other to form complementary regions, as described herein.

[0122] As used herein, the term “double strand” for nucleic acids or oligonucleotides refers to a structure formed by complementary base pairing of two antiparallel nucleotide sequences (i.e., in opposite directions), whether formed by two separate nucleic acid chains or by a single folded chain (e.g., via a hairpin).

[0123] "Effective dose" refers to the amount necessary to achieve the desired therapeutic outcome (for a given period of time and for the means of administration). The effective dose of a protein or conjugate can vary depending on factors such as an individual's disease state, age, sex, and weight, as well as the ability of the protein or conjugate to elicit the desired response in the individual. The effective dose is also the amount in which any toxic or adverse effects of the protein or conjugate are outweighed by the beneficial therapeutic effect.

[0124] As used herein, the term "Fc region" refers to a polypeptide that contains the CH2 and CH3 domains of a constant region of an immunoglobulin (e.g., IgG1, IgG2, IgG3, or IgG4). Optionally, the Fc region may comprise a portion or the entire hinge region of an immunoglobulin (e.g., IgG1, IgG2, IgG3, or IgG4). In some embodiments, the Fc region is a human IgG Fc region, such as the human IgG1 Fc region, human IgG2 Fc region, human IgG3 Fc region, or human IgG4 Fc region. In some embodiments, the Fc region is a modified IgG Fc region having reduced or eliminated effector functions compared to the corresponding wild-type IgG Fc region. The residue numbering in the Fc region is based on the EU index as described in Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Bethesda, MD: US Dept. of Health and Human Services, Public Health Service, National Institutes of Health, 1991). The boundaries of the Fc region of the immunoglobulin heavy chain can vary, and the human IgG heavy chain Fc region is generally defined as the segment from the N-terminus of the CH2 domain (e.g., amino acid residue at position 231 according to the EU index number) to the C-terminus of the CH3 domain (or the C-terminus of the immunoglobulin).

[0125] The term "knockdown" or "expression knockdown" refers to the reduction in the expression of a gene or target mRNA or protein after treatment with reagents.

[0126] As used herein, "modified internucleotide bond" means an internucleotide bond with one or more chemical modifications compared to a reference internucleotide bond having a phosphodiester bond. The modified internucleotide bond can be a non-naturally occurring bond. In some embodiments, the modified internucleotide bond is a phosphate thioester bond.

[0127] As used herein, a “modified nucleotide” means a nucleotide having one or more chemical modifications compared to the corresponding reference nucleotide selected from: adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, adenine deoxyribonucleotide, guanine deoxyribonucleotide, cytosine deoxyribonucleotide, and thymidine deoxyribonucleotide. A modified nucleotide may have one or more chemical modifications, for example, in its sugar, nucleotide, and / or phosphate groups. Alternatively or additionally, a modified nucleotide may have one or more chemical moieties conjugated to the corresponding reference nucleotide. In some embodiments, the modified nucleotide is a 2'-fluorinated nucleotide, a 2'-O-methylated nucleotide, a 2'-deoxynucleotide (DNA), or a 2'-O-alkylated nucleotide. In some embodiments, the modified nucleotide has a phosphate analog, such as 5'-vinylphosphonate. In some embodiments, the modified nucleotide has a debasement moiety or a reverse debasement moiety, such as those shown in Table 6.

[0128] As used herein, "nucleotide" refers to an organic compound having a nucleoside (nucleobase, such as adenine, cytosine, guanine, thymine, or uracil, linked to a phosphate group, and a pentose, such as ribose or 2'-deoxyribose) attached to it. A "nucleotide" can serve as a monomeric unit of nucleic acid polymers such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0129] As used in this article, "empty arm" means an antibody arm that does not bind to any known human target.

[0130] As used herein, "oligonucleotide" means a polymer of linked nucleotides, each of which may be modified or unmodified. Oligonucleotides are typically less than about 100 nucleotides in length.

[0131] As used herein, "protrusion" refers to one or more unpaired nucleotides that protrude from the double-stranded structure of a double-stranded oligonucleotide. A protrusion may include one or more unpaired nucleotides extending from the double-stranded region at the 5' or 3' end of the double-stranded oligonucleotide. A protrusion may be a 3' or 5' protrusion on the antisense or sense strand of the double-stranded oligonucleotide.

[0132] As used in this article, the term "patient" refers to a person who is a patient.

[0133] As used herein, "phosphate analog" means a chemical moiety that mimics the electrostatic and / or steric properties of a phosphate group. In some embodiments, the phosphate analog is located at the 5' end of the oligonucleotide, replacing the 5'-phosphate, which is sometimes susceptible to enzymatic removal. The 5' phosphate analog may include phosphatase-resistant binding. Examples of phosphate analogs include 5'-methylenephosphonate (5'-MP) and 5'-(E)-vinylphosphonate (5'-VP). In some embodiments, the phosphate analog is 5'-VP.

[0134] The term "% sequence identity" or "percentage sequence identity" in relation to a reference nucleic acid sequence is defined as the percentage of nucleotides, nucleosides, or nucleobases in a candidate sequence that are identical to those in the reference nucleic acid sequence after optimal alignment and the introduction of gaps or overhangs where necessary to achieve maximum percentage sequence identity. Alignments used to determine percentage nucleic acid sequence identity can be performed in various ways within the art, for example, using publicly available computer software programs such as those described in Current Protocols in Molecular Biology (Ausubel et al., eds., 1987, Supplement 30, Section 7.7.18, Table 7.7.1), and including BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), Clustal W2.0, or Clustal X2.0 software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences to be compared. The percentage of "sequence identity" can be determined by comparing two best-aligned sequences in a comparison window, where the nucleic acid sequence fragments in the comparison window may contain additions or deletions (e.g., gaps or overhangs) compared to the reference sequence (which does not contain additions or deletions). The percentage can be calculated by determining the number of positions in both sequences where the same nucleotide, nucleoside, or nucleotide appears, to obtain the number of matching positions; dividing the number of matching positions by the total number of positions in the comparison window; and multiplying the result by 100 to obtain the percentage of sequence identity. The output is the percentage identity of the subject sequence relative to the query sequence.

[0135] As used herein, the terms “peptide” or “protein” refer to a polymer of amino acid residues. This term applies to polymers containing naturally occurring amino acids and polymers containing one or more non-naturally occurring amino acids.

[0136] As used herein, “RNAi,” “RNAi reagent,” “iRNA,” “iRNA reagent,” or “RNA interference reagent” means a reagent that mediates the sequence-specific degradation of a target mRNA through RNA interference, such as via the RNA-induced silencing complex (RISC) pathway. In some embodiments, the RNAi reagent has a sense strand and an antisense strand, and the sense strand and antisense strand form a double helix (e.g., double-stranded RNA).

[0137] As used herein, a “chain” refers to a single sequence of adjacent nucleotides linked together by internucleotide bonds (e.g., phosphodiester bonds or thiophosphate bonds). A chain may have two free ends (e.g., a 5' end and a 3' end).

[0138] As used herein, “treatment” or “treating” refers to all processes that may involve the reduction, control, delay, or cessation of the symptoms or progression of the condition or disease disclosed herein, or the improvement of the symptoms or conditions of the condition or disease, but does not necessarily indicate the complete elimination of all symptoms or conditions of the condition or disease. Treatment includes the administration of proteins or nucleic acids or carriers or compositions for the treatment of a patient, particularly a disease or condition in a person.

[0139] The following examples are provided to illustrate, but not to limit, the invention. Example

[0140] Example 1: Generation and characterization of TfR-binding proteins Production of human TfR-binding protein Antibodies against human TfR were generated by immunizing AlivaMab® transgenic mice with the extracellular domain of the His-tagged human transferrin receptor 1 protein (hTfR-ECD-6His, SEQ ID NO: 170, see Table 8) and the mouse transferrin receptor protein (mTfR, SEQ ID NO: 169). Antigen-positive B cells were sorted from the confluent spleen. The binding of the various antibodies cloned from these B cells to the His-tagged hTfR-ECD was verified.

[0141] Additional antibodies against human TfR were generated by immunizing AlivaMab® transgenic mice with the top domain of the His-tagged human transferrin receptor 1 protein (hTfR-ApD-6His, SEQ ID NO: 171, see Table 8). Antigen-positive B cells were sorted from the confluent spleen. The binding of the individual antibodies cloned from these B cells to the His-tagged hTfR-ECD was verified.

[0142] Table 8. Sequences of immunogens used to generate human or mouse TfR antibodies.

[0143] Affinity variants of the resulting human TfR antibodies were prepared by systematically introducing mutations into each CDR of each antibody, and the resulting variants were subjected to multiple rounds of selection with reduced antigen concentration and / or increased dissociation time to isolate clones with improved affinity. The sequences of each variant were used to construct combinatorial libraries, which were then subjected to another round of selection with increased stringency to identify additional or co-mutant pairings between the individual CDR regions. The combinatorial clones were sequenced. Table 1a provides the heavy and light chain CDR and VH / VL sequences of the human TfR binding domain and protein.

[0144] Human TfR-binding proteins are generated using recombinant DNA technology. These proteins can be expressed in mammalian cell lines such as HEK293 or CHO, using an expression system with an optimal predetermined HC:LC vector ratio or a single vector system encoding both HC and LC, for transient or stable transfection. The clarified culture medium into which the protein has been secreted can be purified using common techniques.

[0145] Combining affinity The binding affinity and binding stoichiometry of the exemplary human TfR-binding protein to human and cynomolgus monkey TfR were characterized using surface plasmon resonance assays on a Biacore 8K instrument pretreated with HBS-EP+ (10 mM Hepes pH 7.4 + 150 mM NaCl + 3 mM EDTA + 0.05% (w / v) surfactant P20) run buffer and set at an analysis temperature of 37 °C. Target human and cynomolgus monkey TfR ECDs were immobilized onto a CM4 chip (Cytiva P / N29104989) using standard NHS-EDC amine coupling. The TfR-binding protein was prepared at final concentrations of 0.3, 0.1, 0.033, 0.01, 0.0033, 0.001, 0.00033, and 0.0001 µM by diluting the stock solution into the run buffer.

[0146] Binding analysis was performed using a multi-cycle kinetic approach. Each analysis cycle consisted of the following: (1) injecting antibody or protein at 50 μL / min into all Fc cells from lowest to highest concentration for 140 seconds, followed by a 400-second return buffer flow to monitor the dissociation phase; (2) injecting 3M magnesium chloride at 100 µL / min into all cells for 30 seconds to regenerate the chip surface; and (3) equilibrating the chip surface with 50 µL (30 seconds) of HBS-EP+. Data were processed using a standard dual-reference method and fitted to a two-state binding model using Biacore 8K evaluation software to determine the binding rate (k). on M-1 s -1 Unit), dissociation rate (k off s -1 (unit) and R max (RU unit). Equilibrium dissociation constant (K) D From relation K D = k off / k on Perform the calculations, and express the results in moles. The results are provided in Table 9.

[0147] Table 9. Binding affinity of human TfR-binding proteins for human or cynomolgus monkey TfR at 37°C .

[0148] Example 2: Synthesis and characterization of dsRNA targeting APP Single strands (sense and antisense) of dsRNA duplexes are typically synthesized on a solid support using an automated oligonucleotide synthesizer such as the MerMade™ 12 (LGC Biosearch Technologies) or similar instrument. The sequences of the sense and antisense strands are shown in Tables 5a or 5b. The sense strand is synthesized using a suitable CPG, such as 3'-cholesterol-TEG CNA CPG 500 (LGC Biosearch Technologies) or phthalamido-amino C6 lcaa CPG 500 Å (Chemgenes), while the antisense strand uses a standard support (LGC Biosearch Technologies). The oligonucleotides are synthesized via phosphoramide chemistry at a scale appropriate for in vitro or in vivo experiments.

[0149] Standard reagents were used in the oligonucleotide synthesis (Table 11), with 0.1 M hydroflavin in pyridine used as a sulfidation agent, and a 20% DEA ACN solution used as a post-synthetic auxiliary detergent. All monomers (Table 12a) were prepared in 0.1 M ACN and contained molecular sieve traps. The structures of the linked cholesterol are shown in Table 12b.

[0150] Antisense strands are typically cleaved and deprotected (C / D) at 45°C for 16–24 hours. Sense strands are typically cleaved and deprotected from CPG at ambient temperature using a cold 50% (methylamine / ammonium hydroxide 28–30%) solution for 2–3 hours, while a 3% DEA solution of ammonium hydroxide (28–30%, cold) is typically used for antisense strands. C / D determination is performed by IP-RPLC / MS once the obtained quality data confirms sequence identity. Desilylation of RNA hydroxyl groups can be performed using a DMSO solution of triethylamine trifluoride. Depending on scale, CPG is filtered via a 0.45 μm PVDF syringeless filter, a 0.22 μm PVDF Steriflip® vacuum filter, or a 0.22 μm PVDF Stericup® Quick release. CPG is typically backwashed / rinsed with 30% EtOH / RNase-free water, then filtered through the same filtration apparatus and combined with the first filtrate. This process is repeated twice. The material was then evenly distributed into conical centrifuge tubes for organic matter removal via Genevac™. After concentration, the crude oligonucleotides were diluted back to the synthesis volume with RNase-free water and filtered through a 0.45 µm PVDF syringeless filter, a 0.22 μm PVDF teriflip® vacuum filter, or a 0.22 μm PVDF Stericup® Quick release filter.

[0151] Crude oligonucleotides were purified using anion exchange (AEX) or reversed-phase (RP) chromatography via the AKTA™ Pure purification system. For AEX, an ES Industry Source™ 15Q column was used with MPA: 20 mM NaH₂PO₄, 15% ACN, pH 7.4 and MPB: 20 ​​mM NaH₂PO₄, 1 M NaBr, 15% ACN, pH 7.4. For RP, an ES Industry Source™ 15RPC column was used with MPA: 50 mM sodium acetate, 10% ACN and MPB: 80% ACN. Fractions containing a purity greater than 85% and free of impurities >5% were combined.

[0152] The purified oligonucleotides were desalted using 15 mL 3K MWCO centrifuges at 3500 x g for ~30 min. The oligonucleotides were washed with RNase-free water until the eluent conductivity reached <100 usemi / cm. After desalting, 2–3 mL of RNase-free water was added, and the residue was transferred to a 50 mL Falcon tube by aspiration at 10x. This process was repeated until complete transfer of the oligonucleotides was confirmed by measuring the concentration of the compound on the filter via a nanodrop. The final oligonucleotides were then subjected to 2x nanofiltration at 3500 x g for 2 min using 15 mL 100K MWCO centrifuges. Cholesterol-linked oligonucleotides were annealed at this stage by mixing equimolar aliquots of the sense and antisense strands at room temperature for 30 min to yield cholesterol-conjugated dsRNA. The final desalted oligonucleotides were analyzed for concentration (nanodrop at A260), characterized for mass purity by IP-RP LC / MS (Table 10), and characterized for UV purity by UPLC.

[0153] Table 10: Exemplary LC / MS Data .

[0154] Table 11: Reagents for Oligonucleotide Synthesis .

[0155] Table 12a. Phosphoramide .

[0156] Table 12b. Connected cholesterol structures .

[0157] Example 3: Generation of APP RNAi reagent Some abbreviations are defined as follows: "ACN" refers to acetonitrile; "aAEX" refers to analytical anion exchange; "APP" refers to amyloid precursor protein; "AS" refers to antisense strand; "CPG" refers to glass with controlled aperture; "DAR" refers to the ratio of drug / siRNA to antibody / protein; "DCM" refers to dichloromethane; "DEA" refers to diethylamine; "DHAA" refers to dehydroascorbic acid; "DMSO" refers to dimethyl sulfoxide; "DMT" refers to dimethoxytriphenylmethyl; "dsRNA" refers to double-stranded ribonucleic acid; "DTT" refers to dithiothreitol; "EtOH" refers to ethanol; "h" refers to hours; "HPLC" refers to high-performance liquid chromatography; "IP-RP" refers to... LCMS refers to ion-pair reversed-phase liquid chromatography-mass spectrometry; LC / MS refers to liquid chromatography-mass spectrometry; LTQ / MS refers to linear ion trap mass spectrometry; "min" refers to minutes; "MW" refers to molecular weight; "MWCO" refers to molecular weight cutoff; "NHS" refers to N-hydroxysuccinimide; "OD" refers to optical density; "PBS" is phosphate-buffered saline; "PEG" refers to polyethylene glycol; "PVDF" refers to polyvinylidene fluoride; "RNAi" refers to RNA interference; "rpm" refers to revolutions per minute. RT-qPCR refers to quantitative reverse transcription polymerase chain reaction; SEC refers to size exclusion chromatography; siRNA refers to small interfering RNA; SMCC refers to succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid ester; SS refers to sense chain; TCO refers to trans-cyclooctene; TfR refers to transferrin receptor; THF refers to tetrahydrofuran; TRIS refers to tris(hydroxymethyl)aminomethane; UPLC refers to ultra-high performance liquid chromatography; and UV refers to ultraviolet light.

[0158] SMCC Functionalization of dsRNA Sodium bicarbonate powder (59 mg, 0.702 mmol) was added to a 50 mL conical tube containing an amino-functionalized sense oligonucleotide SS-APP-AMINO (e.g., SEQ ID 172) attached to the C6 amino chain via a 3' terminal thiophosphate ester, as an aqueous solution (8.42 mL, 0.023 mmol, 19.857 mg / mL). The mixture was briefly vortexed and sonicated to dissolve the bicarbonate. Then, an acetonitrile solution (6.32 mL) of freshly prepared (2,5-dioxopyrrolidone-1-yl)4-[(2,5-dioxopyrrolidone-1-yl)methyl]cyclohexanecarbamate (96 mg, 0.281 mmol) was added to the bicarbonate-oligonucleotide solution, for example, dsRNA-48-PS-C6-amino (8.42 mL, 0.023 mmol, 19.857 mg / mL aqueous solution), and vortexed for 30 seconds. The reaction was then allowed to proceed at ambient temperature with shaking at 300 rpm for 4 hours, during which time the reaction mixture was cooled to 10°C for 15 hours under temperature control on a ThermoMixer® C. At this point, LTQ-MS analysis indicated complete conversion. The reaction was quenched to pH 5 using 1N HCl (621 µL, 0.621 mmol). The quenched reaction mixture was then concentrated to approximately half its volume using a GeneVac™ centrifugal evaporator, and the resulting suspension containing the precipitate was filtered through a 0.22 μm Steri-Flip® instrument to remove the precipitate, followed by a single wash with 5 mL of nuclease-free water. The resulting clear solution containing the oligonucleotide was then diluted to approximately 55 mL with 20% acetonitrile in nuclease-free water and concentrated using a CentriCon® ultrafiltration instrument (3000 MW CO regenerated cellulose membrane). After all volumes have passed through the Centricon®, two additional 55 mL aliquots of nuclease-free water containing 20% ​​acetonitrile are passed through the Centricon® to rinse the material, followed by a final single pass of 55 mL pure Milli-Q® water to remove residual acetonitrile. The osmotic residue is then recovered by inverting the Centricon® instrument onto the accompanying recovery cup. The Centricon® instrument is then washed and aspirated twice with 800 µL of nuclease-free water in each of the two filter wells (1.6 mL total per wash), and the combined rinsate and osmotic residue are passed through a 50 kWh MWCO filter, which is then rinsed once more with 5 mL of nuclease-free water.Finally, the concentration of the desired compound was measured using a NanoDrop™ instrument (OD260 - calculated extinction coefficient: 216.09 mmol⁻¹cm⁻¹) to yield the desired compound (SEQ ID 172 with attached C6-amino-SMCC) as a 9.77 mg / mL solution in 13.219 mL (129 mg, 68.1%). LTQ-MS: observed deconvolution m / z = 7361.7, calculated mass 7361.17, mass purity 91.37%.

[0159] SMCC-dsRNA duplex To a conical tube containing SS-APP-AMINO-SMCC, such as SEQ ID NO 172 (12.05 mL, 0.016 mmol, 1.328 mmol / L) with attached C6-amino-SMCC, add its corresponding SS-APP-ANTISENSE, such as SEQ ID NO 173 (0.0165 mmol, 2.619 mmol / L) with 5'-E-vinylphosphonate. The solution was shaken at 25°C for 30 min to yield the desired SMCC-functionalized dsRNA (SMCC-dsRNA), and then refrigerated at 10°C for storage. The annealed solution was sampled for LTQ purity and UPLC non-denaturing chromatography. Analysis by non-denaturing UPLC (run at 10°C) showed a major singlet of 92% purity. LTQ-MS: (m / z of observed deconvolution for the antisense chain = 7768.4, computed 7769.04; m / z of observed deconvolution for the sense chain = 7360.4, computed 7361.17).

[0160] Conjugation schemes for SMCC-functionalized dsRNA The conventional conjugation method utilizes SMCC-functionalized dsRNA to conjugate to a modified cysteine ​​residue of a TfR-binding protein. For this method, the TfR-binding protein is prepared similarly to the method described above, allowing the modified thiol to be used for conjugation through reduction and oxidation of the TfR-binding protein. This is then followed by overnight conjugation at 4°C by incubating the SMCC-dsRNA with 4 molar equivalents of the TfR-binding protein.

[0161] Optionally, a maleimide hydrolysis step can be performed after conjugation to ensure the linker-load is in the final stage and to avoid deconjugation during human circulation via reverse Michael addition. This succinimide ring hydrolysis process is accomplished by raising the pH of the conjugate to 9.0 using 50 mM arginine (using a stock solution of 0.7 M arginine, pH 9.0) and incubating the solution at 37°C for 20 hours. The hydrolytic state of the maleimide, which is generated by the addition of water to the succinimide ring, was confirmed by +18 Da LCMS characterization.

[0162] Step 1a: Conjugation with TfR-binding proteins of the SMCC linker .

[0163] Step 1b: Conjugation with TfR-binding proteins using SMCC linkers to open the loop .

[0164] The synthesis of Mal-Tet-TCO and GDM linkers, and the conjugation of Mal-Tet-TCO or GDM linker-functionalized dsRNAs to modified cysteine ​​residues of TfR-binding proteins, have been described in WO 2024 / 036096.

[0165] Conjugation was monitored using analytical anion exchange chromatography. For the following method, a ProPac™ SAX-10 HPLC column (10 µm particle size, 4 mm diameter, 250 mm length) was used. The flow rate was 1 mL / min, with buffer A: 20 mM TRIS pH 7.0 and buffer B: 20 ​​mM TRIS pH 7.0 + 1.5 M NaCl, at 30 °C.

[0166] The drug / siRNA to antibody / protein ratio (DAR) was calculated based on the peak area percentage from analytical anion exchange (aAEX) chromatography.

[0167] Following conjugation of dsRNA to TfR-binding proteins, excess dsRNA and unconjugated proteins are removed through further purification. Preparative size exclusion chromatography (SEC) or preparative anion exchange chromatography is used for the purification of the final conjugate. Preparative SEC is performed under isocratic conditions using Cytiva Superdex® 200 in 1X PBS pH 7.2. Alternatively, anion exchange chromatography, such as ThermoFisher POROS, is used with a starting buffer of 20 mM TRIS pH 7.0 and elution with a 20 column volume gradient of buffer containing 20 mM TRIS pH 7.0 and 1 M NaCl. TMXQ. This resulted in purified TfR-binding protein-dsRNA conjugates containing no excess dsRNA and a minimal amount of unconjugated protein. A profile of the conjugates obtained by analytical anion exchange analysis was used for final DAR quantification (Table 13).

[0168] Table 13. siRNA / drug to TBP / antibody ratio (DAR) .

[0169] Example 4: In vitro characterization of APP RNAi reagent In vitro efficacy evaluation of cholesterol-conjugated APP-targeting dsRNAs in SHSY5Y and mouse cortical neurons The selected APP RNAi reagent (cholesterol-conjugated APP-targeting dsRNA) was tested in vitro for APP inhibition in cultured SH-SY5Y cells and mouse cortical neurons.

[0170] SH-SY5Y Cell Culture, RNAi Treatment, and Analysis: SH-SY5Y cells (ATCC CRL-2266) were derived from the SK-N-SH neuroblastoma cell line (Ross, RA, et al., 1983. J Natl Cancer Inst 71, 741-747). The basal medium consisted of a 1:1 mixture of Eagle's minimum essential medium (catalog number 30-2003) prepared by ATCC and F12 medium. The complete growth medium was supplemented with additives including 10% fetal bovine serum. Cells were incubated at 37°C under a humidified atmosphere of 5% CO2. On day 1, SH-SY5Y cells were plated in fibronectin-coated tissue culture plates and allowed to adhere overnight. On day 2, the complete medium was removed and replaced with RNAi reagent in serum-free medium. Cells were incubated with the RNAi reagent for 72 hours, followed by analysis of gene (mRNA) expression. Following the manufacturer's protocol (ThermoFisher A35377), RT-qPCR was performed using the TaqMan™ Fast Advanced Cell-to-CT kit to quantify targeted mRNA levels. A normalized ΔΔCT method targeting the housekeeping gene GAPDH (ThermoFisher, Hs99999905_m1, GAPDH; Hs00169098_m1, APP) was used to determine the relative levels of gene (mRNA) expression. Three-parameter or four-parameter logistic fitting was used to determine the IC50. 50 .

[0171] Primary mouse cortical neurons (MCNs) culture, RNAi treatment, and analysis: Primary mouse cortical neurons were isolated from wild-type C57BL6 mouse embryos at E18. On day 7, half the culture medium was removed from each well, and 2x concentration of RNAi reagent in medium containing 2% FBS was added, followed by incubation with cells for 7 days. After treatment, RT-qPCR was performed using the TaqMan™ Fast Advanced Cell-to-CT kit to quantify targeted mRNA levels. A normalized ΔΔCT method targeting the housekeeping gene β-actin probes (ThermoFisher, Mm02619580_g1, ACTB; Mm01344172_m1, APP) was used to determine the relative levels of gene (mRNA) expression. Three-parameter or four-parameter logistic fitting was used to determine IC50. 50 .

[0172] As shown in Tables 14A, 14B, and 15, cholesterol-conjugated dsRNAs targeting the APP coding region (Tables 14A and 14B) or the 3'UTR (Table 15) successfully reduced human APP gene (mRNA) expression in SHSY5Y cells and mouse cortical neurons. Table 16 shows the efficacy of cholesterol-conjugated APP-targeting dsRNAs with different 2'-fluorine modification patterns of sense or antisense strands.

[0173] Table 14A: In vitro knockdown (KD) of APP mRNA by targeting cholesterol-conjugated dsRNA of the APP coding sequence.

[0174] Table 14B: In vitro IC50 of APP RNAi reagent.

[0175] Table 15: In vitro knockdown of APP mRNA by cholesterol-conjugated dsRNA targeting the APP 3'UTR.

[0176] Table 16: In vitro knockdown of APP mRNA by cholesterol-conjugated dsRNA with different chemical modification patterns.

[0177] In vitro efficacy evaluation of TfR-binding protein-dsRNA conjugates targeting APP in EFO-21 and mouse cortical neurons The selected APP RNAi reagent (APP-targeting TfR-binding protein-dsRNA conjugate) was tested in vitro for APP inhibition in EFO-21 cells and mouse cortical neurons (MCN).

[0178] EFO-21 Cell Culture, RNAi Treatment, and Analysis: EFO-21 cells (Simon, WE, et al., 1983. JNatl Cancer Inst 70, 839-845) are derived from human ovarian cancer. The basal medium consisted of RPMI supplemented with 20% fetal bovine serum. Cells were incubated at 37°C in a humidified atmosphere with 5% CO2. On day 1, EFO-21 cells were plated in tissue culture plates and allowed to adhere overnight. On day 2, the medium was removed and replaced with medium containing RNAi reagent and 1.5% serum. Cells were incubated with the RNAi reagent for 72 hours, followed by analysis of gene (mRNA) expression. RT-qPCR was performed using the TaqMan™ FastAdvanced Cell-to-CT kit, following the manufacturer's protocol (ThermoFisher A35377), to quantify targeted mRNA levels. A normalized ΔΔCT method for the housekeeping gene GAPDH (ThermoFisher, Hs99999905_m1, GAPDH; Hs00169098_m1, APP) was used to determine the relative levels of gene (mRNA) expression. Three-parameter or four-parameter logistic fitting was used to determine the IC50. 50 .

[0179] Figure 2A-2B The results provided confirm that two human TfR-binding protein-dsRNA conjugates successfully targeted human APP and reduced APP gene (mRNA) expression in EFO-21 cells. The potency of the TfR-binding protein-dsRNA conjugates is equivalent to that of cholesterol-conjugated dsRNA. Binding of TfR to the TfR-binding protein in the conjugate appears to be required for the observed gene silencing, as isotype Ab-APP dsRNA did not show significant efficacy at any of the drug concentrations tested.

[0180] Mouse cortical neurons and RNAi treatment and analysis. Primary mouse cortical neurons were isolated from wild-type C57BL6 mouse embryos at E18 and cultured as described above. On day 7, half of the culture medium was removed from each well, and 2x concentrations of dsRNA were added in the form of cholesterol- or antibody-conjugated dsRNA (isotype antibody APP siRNA or mTBP1 antibody APP siRNA) in medium containing 2% FBS, and incubated with the cells for 7 days of treatment. After treatment, RT-qPCR was performed using the TaqMan™ Fast Advanced Cell-to-CT kit to quantify targeted mRNA levels. A ΔΔCT method normalized to the housekeeping gene β-actin probes (ThermoFisher, Mm02619580_g1, ACTB; Mm01344172_m1, APP) was used to determine the relative levels of gene (mRNA) expression. Three-parameter or four-parameter logistic fitting was used to determine IC50. 50 .

[0181] Figure 2C and 2D The results provided confirm that two mouse TfR-binding protein (mTBP1)-dsRNA conjugates successfully targeted mouse APP and reduced APP gene (mRNA) expression in primary mouse cortical neurons. The potency of the mTBP1-dsRNA conjugates is similar to that of cholesterol-conjugated dsRNAs. Figure 2C-2D The mTfR-binding protein (mTBP1)-APPdsRNA conjugate showed IC 50 More than 30 times better than the same type of Ab-APP dsRNA ( Figure 2C and 2D Overall, these results support the idea that TfR combined with Ab enhances the efficacy of APP-siRNA in therapeutically targeted cell populations, including neurons.

[0182] Example 5: In vivo characterization of APP RNAi reagent Evaluation of the in vivo potency of cholesterol-conjugated APP-targeting dsRNA in mice after intraventricular (ICV) dose. The selected APP RNAi reagent (cholesterol-conjugated dsRNA targeting APP) was also studied in wild-type C57BL / 6N mice. Mice received ICV injections of 30 µg of APP RNAi reagents with different 2'-fluorine modification patterns (dsRNAs No. 48 and No. 63 in Table 7a) or PBS (phosphate-buffered saline) and were sacrificed on day 14 post-injection. Mouse APP mRNA expression in the brain was measured and analyzed by qPCR using the APP probe (Mm00431829_m1). The standardized ΔΔCT assays used included housekeeping genes, β-actin, and GAPDH probes (Mm02619580_g1 and Mm99999915_g1, respectively). Protein expression was quantified using immunoassays to evaluate the levels of Aβ(1-x), Aβ(1-40), and Aβ(1-42) peptides in homogenized brain tissue. In short, proteins for Aβ peptide analysis were extracted from brain tissue using a guanidine-HCl extraction protocol that captures both soluble and insoluble Aβ species. The assay for detecting Aβ(1-x) protein in brain homogenates was a standard sandwich enzyme-linked immunosorbent assay (ELISA) using commercially available or in-house generated antibody and protein standards. Specifically, the capture antibody used was M266 (Haraln / Envigo), which recognizes epitopes 13-28 of the Aβ(1-42) peptide aa. The detection antibody was an in-house generated biotinylated mouse-specific Aβ(1-42) peptide aa 1-5 epitope antibody. The recombinant protein standard was rodent (rat) Aβ(1-42). The ELISA assay was performed using UltraTMB-ELISA substrate (Thermo Scientific). Analytical data were normalized to the total protein concentration of the brain sample and reported as pg / mg brain.

[0183] Figure 3A and 3B The results shown illustrate the efficacy of the tested APP RNAi reagent 7 days after a single ICV dose. Figure 3A Both APP RNAi agents showed that they reduced the expression of the mouse APP gene (mRNA) in AD-related brain regions, such as the hippocampus and prefrontal cortex. Figure 3A ) and protein expression level ( Figure 3B ). Figure 3B This study confirmed a reduction in β-amyloid (Aβ) peptide (generated via secretase cleavage of the APP protein), which aggregates and forms the matrix of extracellular amyloid plaques found in the brain tissue of individuals with Alzheimer's disease (AD), Down syndrome, and cerebral amyloid angiopathy (CAA). Additionally, Figure 3A and 3BThe effects of different 2'-fluorine modification patterns of the APP RNAi reagent on the efficacy of APP gene silencing were demonstrated.

[0184] In vivo efficacy evaluation of APP-targeting TfR-binding protein-dsRNA conjugates in mice and cynomolgus monkeys following a single peripheral IV dose.

[0185] To confirm that TfR-binding protein-dsRNA conjugates cross the blood-brain barrier (BBB) ​​to deliver dsRNA payloads to the CNS, selected APP RNAi reagents were investigated to evaluate pharmacokinetic efficacy and corresponding brain exposure following peripheral delivery via intravenous route. Specifically, human TfR knock-in mice, in which the extracellular domain of the transferrin receptor had been humanized, received a single 10 mg / kg (dsRNA) IV dose of the APP-targeting human TfR-binding protein-dsRNA conjugate TBP4-dsRNA number 48 (DAR2) or TBP5-sdRNA number 48 (DAR1), or a PBS (phosphate-buffered saline) control. Animals were sacrificed 28 days post-injection. Brain samples were collected to evaluate pharmacokinetic efficacy and tissue exposure. To measure brain tissue exposure, time points between 0.25 and 672 hours (28 days) post-dose were collected, and conjugate-associated dsRNA levels (ng / g) were quantified by reverse-phase LC / MS (IP RP LC / MS) after antibody enrichment via immunoprecipitation.

[0186] The results are shown in Figure 4A and 4B A single IV administration of the APP RNAi reagent resulted in decreased APP mRNA levels in disease-associated cortical and hippocampal regions in mice. Figure 4A To understand the effects of DAR on efficacy and brain exposure, head-to-head dosing was performed on TBP5-dsRNA conjugate number 48 (DAR1) and TBP4-dsRNA conjugate number 48 (DAR2) in a comparative study. Figure 4A The results showed that the TBP5-dsRNA conjugate number 48 (DAR1) reduced mouse APP mRNA levels in the prefrontal cortex and hippocampus by 92% and 85%, respectively. The TBP4-dsRNA conjugate number 48 (DAR2) reduced mouse APP mRNA levels in the prefrontal cortex and hippocampus by 82% and 73%, respectively. Based on AUC (0–672 hours), Figure 4B The study showed that the level of DAR1 conjugate-associated dsRNA in brain tissue was 7.2 times that of DAR2 conjugate-associated dsRNA.

[0187] To further confirm the efficacy of TfR shuttle conjugates, in vivo studies of dsRNA conjugates targeting different APPCDS and 3'UTR sequences were also evaluated in hTfR mice. Table 17 reports the reduction in APP mRNA levels in disease-related cortical and hippocampal regions achieved with multiple dsRNA targeting sequences 28 days after a single 1 mg / kg IV bolus injection (mean, n=4).

[0188] Table 17: In vivo knockdown of APP mRNA by TfR-binding protein-dsRNA conjugates targeting the APPCDS and 3'UTR sequences.

[0189] The efficacy of the selected TfR-binding protein-dsRNA conjugate was further tested in cynomolgus monkeys (Macacafascicularis). To evaluate efficacy, cynomolgus monkeys (n=4 per group) received a single injection of 10 mg / kg (effective dsRNA concentration) via the saphenous vein in the thigh. Monkeys were injected with PBS (phosphate-buffered saline) or APP RNAi reagent TBP4-dsRNA number 48 (DAR2) or TBP5-dsRNA number 48 (DAR1) and sacrificed 29 days post-administration. Deeply anesthetized animals underwent cardiac perfusion, after which brain, spinal cord, and peripheral tissues were collected. Coronal sections of the perfused brain were prepared, and perforations were collected from subregions including the prefrontal cortex, temporal cortex, motor cortex, parietal cortex, and hippocampus and frozen. Additional tissues were also collected from the spinal cord, liver, kidneys, and muscles. Target mRNA and protein levels were evaluated in tissue homogenates by RT-qPCR and ELISA, respectively. Human APP mRNA expression levels were quantified using the ΔΔCT method, with GAPDH serving as a housekeeping gene for the CNS region. Furthermore, conjugate-associated dsRNA levels in brain tissue (28-day endpoint) and plasma (various time points between 0 and 672 hours) were evaluated by IP RP LC-MS. Proteins for Aβ peptide analysis were extracted from brain tissue using a guanidine-HCl extraction protocol that captures both soluble and insoluble Aβ species. Assays for detecting Aβ(1-x) protein in brain homogenates were performed using a standard sandwich enzyme-linked immunosorbent assay (ELISA) employing commercially available or in-house generated antibody and protein standards. Briefly, the capture antibody used was M266 (Haraln / Envigo), which recognizes epitopes 13-28 of the Aβ(1-42) peptide aa. The detection antibody for cynomolgus monkeys was an in-house generated biotinylated 3D6 human / cynomolgus monkey Aβ(1-42) peptide aa 1-5 epitope antibody. The recombinant protein standard used for cynomolgus monkeys was human Aβ(1-40). ELISA assays were performed using UltraTMB-ELISA substrate (Thermo Scientific). Analyzed data were normalized to the total protein concentration of the brain sample and reported as pg / mg brain.

[0190] Figure 5A The study showed a reduction in APP mRNA in disease-related hippocampal and cortical regions 28 days after a single IV dose of either TBP4-dsRNA number 48 or TBP5-dsRNA number 48. Treatment with TBP5-dsRNA number 48 resulted in a reduction in APP mRNA in 62% of the hippocampus, 75% of the prefrontal cortex, 72% of the motor cortex, 64% of the parietal cortex, and 69% of the temporal cortex. Figure 5BThe study showed a decrease in Aβ(1-x) protein, including Aβ(1-42) and Aβ(1-40), in key brain regions at day 28 post-dose compared to the PBS-treated control group. The decrease in Aβ protein levels was associated with a decrease in APP mRNA in key tissues at day 28, with a 72% decrease in the hippocampus, a 76% decrease in the prefrontal cortex, a 73% decrease in the motor cortex, a 69% decrease in the parietal cortex, and a 75% decrease in the temporal cortex. Similar decreases were observed with respect to other APP protein processing fragments such as sAPPα (data not shown). Evaluation of peripheral tissues indicated significant decreases in APP mRNA in tissues such as the gastrocnemius muscle and liver, but no significant decreases in the kidneys or spleen. A comparison of TBP4-dsRNA number 48 (DAR2) and TBP5-dsRNA number 48 (DAR1) showed that the TBP5-dsRNA number 48 (DAR1) conjugate was more potent than the TBP4-dsRNA number 48 (DAR2) conjugate. Figures 5A-5B This is related to Figure 4A This is consistent with the single-dose mouse ICV studies shown.

[0191] The evaluation of conjugate-related dsRNA levels is presented in Figure 5C -D. On day 29, the concentrations of the antisense strand of dsRNA in the prefrontal cortex and hippocampus were 80.6 ng / g (n=1) and 104.9 ng / g (mean n=3) for the TBP5-dsRNA number 48 (DAR1) conjugate, and below the detection threshold LLOQ for the TBP4-dsRNA number 48 (DAR2) conjugate. These levels were two orders of magnitude lower than those observed in the spleen, kidney, and heart, and three orders of magnitude lower than those observed in the liver. AUC (0–672 h) showed a 1.7-fold increase in plasma PK exposure for the TBP5-dsRNA number 48 (DAR1) conjugate compared to that for the TBP4-dsRNA number 48 (DAR2) conjugate at the same dsRNA dose. Figure 5D ).

[0192] Figure 6A -D presents another longitudinal study evaluating the persistence (mean ± SEM, n=3) of mRNA and protein reductions following a single 10 mg / kg IV dose of the human TfR-binding protein-dsRNA conjugate, TBP5-dsRNA number 48, in cynomolgus monkeys (3 animals per group). The largest reductions in mRNA and protein were observed at day 29, but persisted for up to 92 days. At day 92, the reduction in APP mRNA was 62% in the prefrontal cortex (…). Figure 6A And in the hippocampus, it was 57% ( Figure 6CAt 92 days, the reduction in Aβ(1-x) protein, including Aβ(1-42) and Aβ(1-40), was 50% in the prefrontal cortex. Figure 6B And in the hippocampus, it is 65% ( Figure 6D ).

[0193] The efficacy of human TfR-binding protein-dsRNA conjugates has been demonstrated via intravenous administration. The efficacy of TBP5-dsRNA conjugate number 48 (DAR1) delivered via a single subcutaneous (SC) administration and its comparison with IV administration were investigated. Figures 7A-7B This study shows head-to-head comparisons of single doses of 3, 1, and 0.3 mg / kg (effective siRNA concentration) delivered via IV or SC administration in hTfR mice. Animals were euthanized under deep anesthesia and underwent cardiac perfusion at day 29, and brain tissue was collected and processed for RT-qPCR. Figure 7A and Figure 7B The results showed that the TBP5-dsRNA conjugate number 48 (DAR1) delivered via IV or SC was similarly highly effective at all evaluated doses, with less than 10% of APP mRNA remaining in the prefrontal cortex. Dose-response in the hippocampus also showed similar efficacy between IV and SC administration routes; in mice administered via SC, 12% of APP mRNA remained at a dose of 3 mg / kg, 18% at a dose of 1 mg / kg, and 36% at a dose of 0.3 mg / kg. These results support the favorable biodistribution of the TBP5-dsRNA conjugate number 48 (DAR1) after subcutaneous delivery to disease-associated brain tissue.

[0194] Example 6: Characterization of APP RNAi reagents containing dsRNA sequences with different chemical modifications The selected APP RNAi reagents with different antisense strand modifications (e.g., dsRNAs numbered 107, 108, 109, and 110 from Table 7a) were tested in vitro in EFO-21 cells for inhibiting APP expression. The EFO-21 cell culture method is described in Example 4.

[0195] The results presented in Table 18 show that cholesterol-conjugated dsRNAs numbered 107, 108, 109, and 110 successfully reduced APP gene (mRNA) expression and its IC50 in EFO-21 cells.

[0196] Table 18: Characterization of dsRNAs with different chemical modifications .

[0197] Selected TfR-binding protein-dsRNA conjugates with different dsRNA modifications were tested in vivo to evaluate pharmacokinetic efficacy following peripheral delivery via intravenous route in both rodents and non-human primates. More specifically, human TfR transgenic knock-in mice, in which the extracellular domain of the transferrin receptor had been humanized, received a single 1 mg / kg (dsRNA) IV dose of TBP5-dsRNA No. 48 (DAR1) or TBP5-dsRNA No. 109 (DAR1), or a PBS (phosphate-buffered saline) control. Animals were sacrificed 28 or 84 days after injection. Brain samples were collected to evaluate pharmacokinetic efficacy, such as... Figure 8 As shown in Figure 9. For cynomolgus monkeys (3-4 animals per group), a single injection of 10 mg / kg (effective dsRNA concentration) was administered via the saphenous vein in the thigh. Monkeys were injected with PBS (phosphate-buffered saline) or the APP RNAi reagent TBP5-dsRNA number 109 (DAR1) and sacrificed 29 or 85 days post-administration. Coronal sections of the perfused brain were prepared, and perforations were collected from subregions including the prefrontal cortex, temporal cortex, motor cortex, parietal cortex, and hippocampus to evaluate pharmacokinetic efficacy, as shown in Figure 9. mRNA and protein expression levels were quantified using the method described in Example 5.

[0198] Figure 8 The study demonstrated a reduction in APP mRNA in disease-related hippocampal and cortical regions at 28 and 84 days following a single IV dose of either TBP5-dsRNA number 48 or TBP5-dsRNA number 109 in hTfR mice. Treatment with TBP5-dsRNA number 48 resulted in a 79% reduction in APP mRNA in the hippocampus and an 87% reduction in the prefrontal cortex at day 28. Furthermore, 3-month durability of TBP5-dsRNA number 48 treatment was observed, with a 60% reduction in APP mRNA in the hippocampus and a 63% reduction in the prefrontal cortex. TBP5-dsRNA number 109, with a reverse debasement cap at the 3' end of the antisense strand, showed an 80% reduction in APP mRNA in the hippocampus and an 85% reduction in the prefrontal cortex at day 28. TBP5-dsRNA number 109 also showed comparable persistence, with a 54% reduction in APP mRNA in the hippocampus and a 61% reduction in the prefrontal cortex 3 months after dose administration.

[0199] Figure 9A-B presents the persistence of mRNA and protein reductions following a single 10 mg / kg IV dose in cynomolgus monkeys (3-4 animals per group) for the human TfR-binding protein-dsRNA conjugate, TBP5-dsRNA number 109. The largest reductions in mRNA and protein were observed at day 29, but persisted for up to 92 days across disease-associated cortical and hippocampal regions. The mean reduction in APP mRNA across these regions of interest was 53% (range 42%-57%) at day 29 and persisted with a mean reduction of 43% (range 38%-49%) at day 85. Figure 9A The average reduction of Aβ(1-x) proteins across these regions of interest, including Aβ(1-42) and Aβ(1-40), was 61% (range 50%–67%) at 29 days and remained at an average reduction of 57% (range 49%–64%) at 85 days. Figure 9B These results confirm that modifications such as the inclusion of a reverse debase cap at the 3' end of the antisense strand, TBP5-dsRNA number 109, achieve robust and durable knockdown in disease-associated brain tissue.

[0200] sequence list .

Claims

1. An APP RNAi reagent comprising formula (I): (RL) n -P, R is a double-stranded RNA (dsRNA) containing a sense strand and an antisense strand, wherein the antisense strand is complementary to the APP mRNA. Where P is a protein containing a monovalent human TfR binding domain; and Where L is a connector, or optionally not present. The human TfR binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 3, LCDR1 comprises SEQ ID NO: 4, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 6; and Where n is an integer from 1 to 3.

2. The APP RNAi reagent according to claim 1, wherein n is 1.

3. The APP RNAi reagent according to claim 1, wherein n is 2.

4. The APP RNAi reagent according to any one of claims 1-3, wherein VH comprises SEQ ID NO: 7 and VL comprises SEQ ID NO:

8.

5. The APP RNAi reagent according to any one of claims 1-4, wherein the human TfR binding domain is Fab, scFv, Fv or scFab.

6. The APP RNAi reagent according to any one of claims 1-5, wherein the human TfR binding domain further comprises a heavy chain constant region containing a cysteine ​​residue at residue 124 (according to EU index number).

7. The APP RNAi reagent according to any one of claims 1-6, wherein the human TfR binding domain further comprises a light chain constant region containing a cysteine ​​residue at residue 156 (according to EU index number).

8. The APP RNAi reagent according to any one of claims 1-7, wherein P further comprises a half-life extender.

9. The APP RNAi reagent according to claim 8, wherein the half-life extender is an immunoglobulin Fc region or a VHH bound to human serum albumin (HSA).

10. The APP RNAi reagent according to claim 9, wherein the half-life extension is an immunoglobulin Fc region.

11. The APP RNAi reagent according to claim 10, wherein the immunoglobulin Fc region is a modified human IgG4 Fc region.

12. The APP RNAi reagent of claim 11, wherein the modified human IgG4 Fc region comprises proline at residue 228 and alanine at residues 234 and 235 (all residues are numbered according to EU index numbers).

13. The APP RNAi reagent according to any one of claims 10-12, wherein P comprises the immunoglobulin Fc region, which contains a cysteine ​​residue at residue 378 (according to the EU index number).

14. The APP RNAi reagent according to any one of claims 10-13, wherein the immunoglobulin Fc region comprises: (a) A first Fc CH3 domain comprising serine at position 349, methionine at position 366, tyrosine at position 370, and valine at position 409; and a second Fc CH3 domain comprising glycine at position 356, aspartic acid at position 357, glutamine at position 364, and alanine at position 407 (all residues are numbered according to EU index numbers); or (b) The first Fc CH3 domain, which contains leucine at residue 405; and the second Fc CH3 domain, which contains arginine at residue 409 (all residues are numbered according to EU index number).

15. The APP RNAi reagent according to any one of claims 1-7, wherein P comprises a heavy chain (HC) and a light chain (LC), wherein HC comprises SEQ ID NO: 9 and LC comprises SEQ ID NO:

10.

16. The APP RNAi reagent according to any one of claims 1-14, wherein P comprises two heavy chains HC1 and HC2 and one light chain LC1, wherein HC1 comprises SEQ ID NO: 14, LC1 comprises SEQ ID NO: 10, and HC2 comprises SEQ ID NO:

15.

17. The APP RNAi reagent according to any one of claims 1-14, wherein P comprises two heavy chains HC1 and HC2 and one light chain LC1, wherein HC1 comprises SEQ ID NO: 16, LC1 comprises SEQ ID NO: 10, and HC2 comprises SEQ ID NO:

17.

18. The APP RNAi reagent according to claim 9, wherein the half-life extender is a VHH bound to HSA.

19. The APP RNAi reagent according to claim 18, wherein the VHH comprises CDR1 comprising SEQ ID NO: 20, CDR2 comprising SEQ ID NO: 21, and CDR3 comprising SEQ ID NO:

22.

20. The APP RNAi reagent according to claim 18 or 19, wherein the VHH comprises SEQ ID NO:

23.

21. The APP RNAi reagent according to any one of claims 18-20, wherein P comprises a heavy chain (HC) and a light chain (LC), and wherein the HC comprises SEQ ID NO: 11 and the LC comprises SEQ ID NO: 12 or 10.

22. The APP RNAi reagent according to any one of claims 1-14, wherein P is a heterodimeric antibody comprising a first arm containing a monovalent human TfR binding domain and a second arm which is an empty arm.

23. The APP RNAi reagent of claim 22, wherein the second arm comprises a heavy chain (HC) and a light chain (LC), and wherein the HC comprises SEQ ID NO: 18 and the LC comprises SEQ ID NO:

19.

24. The APP RNAi reagent according to claim 22 or 23, wherein P comprises two heavy chains HC1 and HC2 and two light chains LC1 and LC2, wherein HC1 comprises SEQ ID NO: 13, LC1 comprises SEQ ID NO: 10, HC2 comprises SEQ ID NO: 18, and LC2 comprises SEQ ID NO:

19.

25. The APP RNAi reagent according to any one of claims 1-24, wherein when L is present, L is a Mal-Tet-TCO adapter, an SMCC adapter, or a GDM adapter.

26. The APP RNAi reagent according to any one of claims 1-25, wherein L is an SMCC adapter.

27. The APP RNAi reagent according to any one of claims 1-26, wherein P is ligated to the 3' end of the sense strand of the dsRNA, said ligation optionally via a adapter.

28. The APP RNAi reagent according to any one of claims 1-27, wherein the sense strand and antisense strand comprise a pair of nucleic acid sequences selected from: (a) The sense chain contains SEQ ID NO: 35, and the antisense chain contains SEQ ID NO: 36; (b) The sense chain contains SEQ ID NO: 37, and the antisense chain contains SEQ ID NO: 38; (c) The sense chain contains SEQ ID NO: 39, and the antisense chain contains SEQ ID NO: 40; (d) The sense chain contains SEQ ID NO: 41, and the antisense chain contains SEQ ID NO: 42; (e) The sense chain contains SEQ ID NO: 43, and the antisense chain contains SEQ ID NO: 44; (f) The sense chain contains SEQ ID NO: 45, and the antisense chain contains SEQ ID NO: 46; (g) The sense chain contains SEQ ID NO: 47, and the antisense chain contains SEQ ID NO: 48; (h) The sense chain contains SEQ ID NO: 49, and the antisense chain contains SEQ ID NO: 50; (i) The sense chain contains SEQ ID NO: 51, and the antisense chain contains SEQ ID NO: 52; (j) The sense chain contains SEQ ID NO: 53, and the antisense chain contains SEQ ID NO: 54; (k) The sense chain contains SEQ ID NO: 55, and the antisense chain contains SEQ ID NO: 56; (l) The sense chain contains SEQ ID NO: 57, and the antisense chain contains SEQ ID NO: 58; (m) The sense chain contains SEQ ID NO: 59, and the antisense chain contains SEQ ID NO: 60; (n) The sense chain contains SEQ ID NO: 61, and the antisense chain contains SEQ ID NO: 62; (o) The sense chain contains SEQ ID NO: 63, and the antisense chain contains SEQ ID NO: 64; (p) The sense chain contains SEQ ID NO: 65, and the antisense chain contains SEQ ID NO: 66; (q) The sense chain contains SEQ ID NO: 67, and the antisense chain contains SEQ ID NO: 68; (r) The sense chain contains SEQ ID NO: 69, and the antisense chain contains SEQ ID NO: 70; (s) The sense chain contains SEQ ID NO: 71, and the antisense chain contains SEQ ID NO: 72; (t) The sense chain contains SEQ ID NO: 73, and the antisense chain contains SEQ ID NO: 74; (u) The sense chain contains SEQ ID NO: 75, and the antisense chain contains SEQ ID NO: 76; (v) The sense chain contains SEQ ID NO: 77, and the antisense chain contains SEQ ID NO: 78; (w) The sense chain contains SEQ ID NO: 79, and the antisense chain contains SEQ ID NO: 80; (x) The sense chain contains SEQ ID NO: 81, and the antisense chain contains SEQ ID NO: 82; (y) The sense chain contains SEQ ID NO: 83, and the antisense chain contains SEQ ID NO: 84; (z) The sense chain contains SEQ ID NO: 85, and the antisense chain contains SEQ ID NO: 86; (aa) The sense chain contains SEQ ID NO: 87, and the antisense chain contains SEQ ID NO: 88; (bb) The sense chain contains SEQ ID NO: 89, and the antisense chain contains SEQ ID NO: 90; (cc) The sense chain contains SEQ ID NO: 91, and the antisense chain contains SEQ ID NO: 92; (dd) The sense chain contains SEQ ID NO: 93, and the antisense chain contains SEQ ID NO: 94; (ee) The sense chain contains SEQ ID NO: 95, and the antisense chain contains SEQ ID NO: 96; (ff) The sense chain contains SEQ ID NO: 97, and the antisense chain contains SEQ ID NO: 98; (gg) The sense chain contains SEQ ID NO: 99, and the antisense chain contains SEQ ID NO: 100; (hh) The sense chain contains SEQ ID NO: 184, and the antisense chain contains SEQ ID NO: 36; (ii) The sense chain contains SEQ ID NO: 188, and the antisense chain contains SEQ ID NO: 38; (jj) The sense chain contains SEQ ID NO: 35, and the antisense chain contains SEQ ID NO: 214; Optionally, one or more nucleotides of the sense strand and antisense strand are independently modified nucleotides, and the bonding between one or more nucleotides of the sense strand and antisense strand is a modified nucleotide bonding.

29. The APP RNAi reagent of claim 28, wherein the sense strand comprises SEQ ID NO: 35 and the antisense strand comprises SEQ ID NO:

36.

30. The APP RNAi reagent of claim 28, wherein the sense strand comprises SEQ ID NO: 35 and the antisense strand comprises SEQ ID NO:

214.

31. The APP RNAi reagent according to claim 28, wherein the sense strand comprises SEQ ID NO: 37 and the antisense strand comprises SEQ ID NO:

38.

32. The APP RNAi reagent according to any one of claims 1-31, wherein one or more nucleotides of the sense strand are modified nucleotides.

33. The APP RNAi reagent of claim 32, wherein each nucleotide of the sense strand is a modified nucleotide.

34. The APP RNAi reagent according to any one of claims 1-33, wherein one or more nucleotides of the antisense strand are modified nucleotides.

35. The APP RNAi reagent of claim 34, wherein each nucleotide of the antisense strand is a modified nucleotide.

36. The APP RNAi reagent according to any one of claims 32-35, wherein the modified nucleotide is a 2'-fluorinated nucleotide, a 2'-O-methyl nucleotide, a 2'-deoxynucleotide (DNA), or a 2'-O-alkyl nucleotide.

37. The APP RNAi reagent according to any one of claims 32-36, wherein the sense strand has four 2'-fluorinated nucleotides at positions 7, 9, 10 and 11 at a distance from the 5' end of the sense strand.

38. The APP RNAi reagent according to claim 37, wherein the nucleotides at positions other than positions 7, 9, 10 and 11 of the sense strand are 2'-O-methyl modified nucleotides.

39. The APP RNAi reagent according to any one of claims 32-38, wherein the antisense strand has four 2'-fluorinated nucleotides at positions 2, 6, 14 and 16 from the 5' end of the antisense strand.

40. The APP RNAi reagent of claim 39, wherein the nucleotides at positions other than positions 2, 6, 14 and 16 on the antisense strand are 2'-O-methyl modified nucleotides.

41. The APP RNAi reagent according to any one of claims 32-36, wherein the sense strand has three 2'-fluorinated nucleotides at positions 9, 10, and 11 at a distance from the 5' end of the sense strand.

42. The APP RNAi reagent according to claim 41, wherein the nucleotides at positions other than positions 9, 10 and 11 of the sense strand are 2'-O-methyl modified nucleotides.

43. The APP RNAi reagent according to any one of claims 32-36, wherein the antisense strand has five 2'-fluorinated nucleotides at positions 2, 5, 7, 14 and 16 from the 5' end of the antisense strand.

44. The APP RNAi reagent according to claim 43, wherein the nucleotides at positions other than positions 2, 5, 7, 14 and 16 on the antisense strand are 2'-O-methyl modified nucleotides.

45. The APP RNAi reagent according to any one of claims 32-36, wherein the antisense strand has five 2'-fluorinated nucleotides at positions 2, 5, 8, 14 and 16 from the 5' end of the antisense strand.

46. ​​The APP RNAi reagent according to claim 45, wherein the nucleotides at positions other than positions 2, 5, 8, 14 and 16 on the antisense strand are 2'-O-methyl modified nucleotides.

47. The APP RNAi reagent according to any one of claims 32-36, wherein the antisense strand has five 2'-fluorinated nucleotides at positions 2, 3, 7, 14 and 16 from the 5' end of the antisense strand.

48. The APP RNAi reagent according to claim 47, wherein the nucleotides at positions other than positions 2, 3, 7, 14 and 16 on the antisense strand are 2'-O-methyl modified nucleotides.

49. The APP RNAi reagent according to any one of claims 1-48, wherein the sense strand and antisense strand have one or more modified nucleotide bonds.

50. The APP RNAi reagent according to claim 49, wherein the modified internucleotide bond is a phosphate thioester bond.

51. The APP RNAi reagent according to claim 49 or 50, wherein the sense strand has four or five phosphate thioester bonds.

52. The APP RNAi reagent according to any one of claims 49-51, wherein the antisense strand has four or five phosphate thioester bonds.

53. The APP RNAi reagent according to any one of claims 1-52, wherein the antisense strand has a phosphate analog at the 5' end.

54. The APP RNAi reagent according to claim 53, wherein the phosphate analog is 5'-vinylphosphonate.

55. The APP RNAi reagent according to any one of claims 1-54, wherein the sense strand or antisense strand comprises a debasement portion or a reverse debasement portion.

56. The APP RNAi reagent according to any one of claims 1-55, wherein the sense strand and antisense strand comprise a pair of nucleic acid sequences selected from: (a) The sense chain contains SEQ ID NO: 101, and the antisense chain contains SEQ ID NO: 102, 173, 176, 177, 178, 179, 180, 182 or 185; (b) The sense chain contains SEQ ID NO: 103, and the antisense chain contains SEQ ID NO: 104, 175, 189, 190, 191, 192, 194, 195 or 196; (c) The sense chain contains SEQ ID NO: 105, and the antisense chain contains SEQ ID NO: 106; (d) The sense chain contains SEQ ID NO: 107, and the antisense chain contains SEQ ID NO: 108; (e) The sense chain contains SEQ ID NO: 109, and the antisense chain contains SEQ ID NO: 110; (f) The sense chain contains SEQ ID NO: 111, and the antisense chain contains SEQ ID NO: 112; (g) The sense chain contains SEQ ID NO: 113, and the antisense chain contains SEQ ID NO: 114; (h) The sense chain contains SEQ ID NO: 115, and the antisense chain contains SEQ ID NO: 116; (i) The sense chain contains SEQ ID NO: 117, and the antisense chain contains SEQ ID NO: 118; (j) The sense chain contains SEQ ID NO: 119, and the antisense chain contains SEQ ID NO: 120; (k) The sense chain contains SEQ ID NO: 121, and the antisense chain contains SEQ ID NO: 122; (l) The sense chain contains SEQ ID NO: 123, and the antisense chain contains SEQ ID NO: 124; (m) The sense chain contains SEQ ID NO: 125, and the antisense chain contains SEQ ID NO: 126; (n) The sense chain contains SEQ ID NO: 127, and the antisense chain contains SEQ ID NO: 128; (o) The sense chain contains SEQ ID NO: 129, and the antisense chain contains SEQ ID NO: 130; (p) The sense chain contains SEQ ID NO: 131, and the antisense chain contains SEQ ID NO: 132; (q) The sense chain contains SEQ ID NO: 133, and the antisense chain contains SEQ ID NO: 134; (r) The sense chain contains SEQ ID NO: 135, and the antisense chain contains SEQ ID NO: 136; (s) The sense chain contains SEQ ID NO: 137, and the antisense chain contains SEQ ID NO: 138; (t) The sense chain contains SEQ ID NO: 139, and the antisense chain contains SEQ ID NO: 140; (u) The sense chain contains SEQ ID NO: 141, and the antisense chain contains SEQ ID NO: 142; (v) The sense chain contains SEQ ID NO: 143, and the antisense chain contains SEQ ID NO: 144; (w) The sense chain contains SEQ ID NO: 145, and the antisense chain contains SEQ ID NO: 146; (x) The sense chain contains SEQ ID NO: 147, and the antisense chain contains SEQ ID NO: 148; (y) The sense chain contains SEQ ID NO: 149, and the antisense chain contains SEQ ID NO: 150; (z) The sense chain contains SEQ ID NO: 151, and the antisense chain contains SEQ ID NO: 152; (aa) The sense chain contains SEQ ID NO: 153, and the antisense chain contains SEQ ID NO: 154; (bb) The sense chain contains SEQ ID NO: 155, and the antisense chain contains SEQ ID NO: 156; (cc) The sense chain contains SEQ ID NO: 157, and the antisense chain contains SEQ ID NO: 158; (dd) The sense chain contains SEQ ID NO: 159, and the antisense chain contains SEQ ID NO: 160; (ee) The sense chain contains SEQ ID NO: 161, and the antisense chain contains SEQ ID NO: 162; (ff) The sense chain contains SEQ ID NO: 163, and the antisense chain contains SEQ ID NO: 164; (gg) The sense chain contains SEQ ID NO: 165, and the antisense chain contains SEQ ID NO: 166; (hh) The sense chain contains SEQ ID NO: 172, and the antisense chain contains SEQ ID NO: 173; (ii) The sense chain contains SEQ ID NO: 181, and the antisense chain contains SEQ ID NO: 173; (jj) The sense chain contains SEQ ID NO: 174 or 193, and the antisense chain contains SEQ ID NO: 175; (kk) The sense chain contains SEQ ID NO: 172, and the antisense chain contains SEQ ID NO: 177; (ll) The sense chain contains SEQ ID NO: 181, 183 or 186, and the antisense chain contains SEQ ID NO: 102; (mm) The sense chain contains SEQ ID NO: 187, 193 or 197, and the antisense chain contains SEQ ID NO: 104; (nn) The sense chain contains SEQ ID NO: 198, and the antisense chain contains SEQ ID NO: 199; (oo) The sense chain contains SEQ ID NO: 200, and the antisense chain contains SEQ ID NO: 201; (pp) The sense chain contains SEQ ID NO: 202, and the antisense chain contains SEQ ID NO: 203; (qq) The sense chain contains SEQ ID NO: 204, and the antisense chain contains SEQ ID NO: 205; (rr) The sense chain contains SEQ ID NO: 206, and the antisense chain contains SEQ ID NO: 207; (ss) The sense chain contains SEQ ID NO: 208, and the antisense chain contains SEQ ID NO: 209; (tt) The sense chain contains SEQ ID NO: 210, and the antisense chain contains SEQ ID NO: 118; The (uu) sense chain contains SEQ ID NO: 211, and the antisense chain contains SEQ ID NO: 120; (vv) The sense chain contains SEQ ID NO: 212, and the antisense chain contains SEQ ID NO: 122; (ww) The sense chain contains SEQ ID NO: 213, and the antisense chain contains SEQ ID NO: 124; (xx) The sense chain contains SEQ ID NO: 172, and the antisense chain contains SEQ ID NO: 215; (yy) The sense chain contains SEQ ID NO: 172, and the antisense chain contains SEQ ID NO: 216; (zz) The sense chain contains SEQ ID NO: 172, and the antisense chain contains SEQ ID NO: 217; The (aaa) sense chain contains SEQ ID NO: 172, and the antisense chain contains SEQ ID NO: 218; (bbb) The sense chain contains SEQ ID NO: 219, and the antisense chain contains SEQ ID NO: 220; (ccc) The sense chain contains SEQ ID NO: 221, and the antisense chain contains SEQ ID NO: 222; (ddd) The sense chain contains SEQ ID NO: 223, and the antisense chain contains SEQ ID NO: 224; The (eee) sense chain contains SEQ ID NO: 225, and the antisense chain contains SEQ ID NO: 226; (fff) The sense chain contains SEQ ID NO: 227, and the antisense chain contains SEQ ID NO: 228; The (ggg) sense chain contains SEQ ID NO: 229, and the antisense chain contains SEQ ID NO: 230; (hhh) The sense chain contains SEQ ID NO: 231, and the antisense chain contains SEQ ID NO: 232; (iii) The sense chain contains SEQ ID NO: 233, and the antisense chain contains SEQ ID NO: 234; (jjj) has a sense chain containing SEQ ID NO: 235 and an antisense chain containing SEQ ID NO: 236; (kkk) The sense chain contains SEQ ID NO: 237, and the antisense chain contains SEQ ID NO: 238; (lll) The sense chain contains SEQ ID NO: 239, and the antisense chain contains SEQ ID NO: 240; and (mmm) The sense chain contains SEQ ID NO: 241, and the antisense chain contains SEQ ID NO:

242.

57. The APP RNAi reagent according to any one of claims 1-56, wherein the sense strand and antisense strand have a pair of nucleic acid sequences selected from: (a) The sense chain consists of SEQ ID NO: 101, and the antisense chain consists of SEQ ID NO: 102, 173, 176, 177, 178, 179, 180, 182 or 185; (b) The sense chain consists of SEQ ID NO: 103, and the antisense chain consists of SEQ ID NO: 104, 175, 189, 190, 191, 192, 194, 195 or 196; (c) The sense chain consists of SEQ ID NO: 105, and the antisense chain consists of SEQ ID NO: 106; (d) The sense chain consists of SEQ ID NO: 107, and the antisense chain consists of SEQ ID NO: 108; (e) The sense chain consists of SEQ ID NO: 109, and the antisense chain consists of SEQ ID NO: 110; (f) The sense chain consists of SEQ ID NO: 111, and the antisense chain consists of SEQ ID NO: 112; (g) The sense chain consists of SEQ ID NO: 113, and the antisense chain consists of SEQ ID NO: 114; (h) The sense chain consists of SEQ ID NO: 115, and the antisense chain consists of SEQ ID NO: 116; (i) The sense chain consists of SEQ ID NO: 117, and the antisense chain consists of SEQ ID NO: 118; (j) The sense chain consists of SEQ ID NO: 119, and the antisense chain consists of SEQ ID NO: 120; (k) The sense chain consists of SEQ ID NO: 121, and the antisense chain consists of SEQ ID NO: 122; (l) The sense chain consists of SEQ ID NO: 123, and the antisense chain consists of SEQ ID NO: 124; (m) The sense chain consists of SEQ ID NO: 125, and the antisense chain consists of SEQ ID NO: 126; (n) The sense chain consists of SEQ ID NO: 127, and the antisense chain consists of SEQ ID NO: 128; (o) The sense chain consists of SEQ ID NO: 129, and the antisense chain consists of SEQ ID NO: 130; (p) The sense chain consists of SEQ ID NO: 131, and the antisense chain consists of SEQ ID NO: 132; (q) The sense chain consists of SEQ ID NO: 133, and the antisense chain consists of SEQ ID NO: 134; (r) The sense chain consists of SEQ ID NO: 135, and the antisense chain consists of SEQ ID NO: 136; (s) The sense chain consists of SEQ ID NO: 137, and the antisense chain consists of SEQ ID NO: 138; (t) The sense chain consists of SEQ ID NO: 139, and the antisense chain consists of SEQ ID NO: 140; (u) The sense chain consists of SEQ ID NO: 141, and the antisense chain consists of SEQ ID NO: 142; (v) The sense chain consists of SEQ ID NO: 143, and the antisense chain consists of SEQ ID NO: 144; (w) The sense chain consists of SEQ ID NO: 145, and the antisense chain consists of SEQ ID NO: 146; (x) The sense chain consists of SEQ ID NO: 147, and the antisense chain consists of SEQ ID NO: 148; (y) The sense chain consists of SEQ ID NO: 149, and the antisense chain consists of SEQ ID NO: 150; (z) The sense chain consists of SEQ ID NO: 151, and the antisense chain consists of SEQ ID NO: 152; (aa) The sense chain consists of SEQ ID NO: 153, and the antisense chain consists of SEQ ID NO: 154; (bb) The sense chain consists of SEQ ID NO: 155, and the antisense chain consists of SEQ ID NO: 156; (cc) The sense chain consists of SEQ ID NO: 157, and the antisense chain consists of SEQ ID NO: 158; (dd) The sense chain consists of SEQ ID NO: 159, and the antisense chain consists of SEQ ID NO: 160; (ee) The sense chain consists of SEQ ID NO: 161, and the antisense chain consists of SEQ ID NO: 162; (ff) The sense chain consists of SEQ ID NO: 163, and the antisense chain consists of SEQ ID NO: 164; (gg) The sense chain consists of SEQ ID NO: 165, and the antisense chain consists of SEQ ID NO: 166; (hh) The sense chain consists of SEQ ID NO: 172, and the antisense chain consists of SEQ ID NO: 173; (ii) The sense chain consists of SEQ ID NO: 181, and the antisense chain consists of SEQ ID NO: 173; (jj) The sense chain consists of SEQ ID NO: 174 or 193, and the antisense chain consists of SEQ ID NO: 175; (kk) The sense chain consists of SEQ ID NO: 172, and the antisense chain consists of SEQ ID NO: 177; (ll) The sense chain consists of SEQ ID NO: 181, 183 or 186, and the antisense chain consists of SEQ ID NO: 102; (mm) The sense chain consists of SEQ ID NO: 187, 193 or 197, and the antisense chain consists of SEQ ID NO: 104; (nn) The sense chain consists of SEQ ID NO: 198, and the antisense chain consists of SEQ ID NO: 199; (oo) The sense chain consists of SEQ ID NO: 200, and the antisense chain consists of SEQ ID NO: 201; (pp) The sense chain consists of SEQ ID NO: 202, and the antisense chain consists of SEQ ID NO: 203; (qq) The sense chain consists of SEQ ID NO: 204, and the antisense chain consists of SEQ ID NO: 205; (rr) The sense chain consists of SEQ ID NO: 206, and the antisense chain consists of SEQ ID NO: 207; (ss) The sense chain consists of SEQ ID NO: 208, and the antisense chain consists of SEQ ID NO: 209; (tt) The sense chain consists of SEQ ID NO: 210, and the antisense chain consists of SEQ ID NO: 118; The (uu) sense chain consists of SEQ ID NO: 211, and the antisense chain consists of SEQ ID NO: 120; (vv) The sense chain consists of SEQ ID NO: 212, and the antisense chain consists of SEQ ID NO: 122; (ww) The sense chain consists of SEQ ID NO: 213, and the antisense chain consists of SEQ ID NO: 124; (xx) The sense chain consists of SEQ ID NO: 172, and the antisense chain consists of SEQ ID NO: 215; (yy) The sense chain consists of SEQ ID NO: 172, and the antisense chain consists of SEQ ID NO: 216; (zz) The sense chain consists of SEQ ID NO: 172, and the antisense chain consists of SEQ ID NO: 217; The (aaa) sense chain consists of SEQ ID NO: 172, and the antisense chain consists of SEQ ID NO: 218; (bbb) The sense chain consists of SEQ ID NO: 219, and the antisense chain consists of SEQ ID NO: 220; (ccc) The sense chain consists of SEQ ID NO: 221, and the antisense chain consists of SEQ ID NO: 222; (ddd) The sense chain consists of SEQ ID NO: 223, and the antisense chain consists of SEQ ID NO: 224; The (eee) sense chain consists of SEQ ID NO: 225, and the antisense chain consists of SEQ ID NO: 226; (fff) The sense chain consists of SEQ ID NO: 227, and the antisense chain consists of SEQ ID NO: 228; The (ggg) sense chain consists of SEQ ID NO: 229, and the antisense chain consists of SEQ ID NO: 230; (hhh) The sense chain consists of SEQ ID NO: 231, and the antisense chain consists of SEQ ID NO: 232; (iii) The sense chain consists of SEQ ID NO: 233, and the antisense chain consists of SEQ ID NO: 234; The (jjj) sense chain consists of SEQ ID NO: 235, and the antisense chain consists of SEQ ID NO: 236; (kkk) The sense chain consists of SEQ ID NO: 237, and the antisense chain consists of SEQ ID NO: 238; (lll) The sense chain consists of SEQ ID NO: 239, and the antisense chain consists of SEQ ID NO: 240; and (mmm) The sense chain consists of SEQ ID NO: 241, and the antisense chain consists of SEQ ID NO:

242.

58. A pharmaceutical composition comprising the APP RNAi reagent according to any one of claims 1-57 and a pharmaceutically acceptable carrier.

59. A method for treating APP-related neurological disorders in patients with such needs, the method comprising administering to the patient an effective amount of the APP RNAi reagent according to any one of claims 1-57, or the pharmaceutical composition according to claim 58.

60. The method of claim 59, wherein the APP-related neurological disease is selected from Alzheimer's disease, Down syndrome, or cerebral amyloid angiopathy.

61. The method of claim 59 or 60, wherein the APP RNAi reagent is administered intravenously or subcutaneously to the patient.

62. The APP RNAi reagent according to any one of claims 1-57, or the pharmaceutical composition according to claim 58, for use in therapy.

63. The APP RNAi reagent according to any one of claims 1-57, or the pharmaceutical composition according to claim 58, for the treatment of APP-related neurological disorders.

64. An APP RNAi reagent or pharmaceutical composition for use according to claim 63, wherein the APP-related neurological disease is selected from Alzheimer's disease, Down syndrome, or cerebral amyloid angiopathy.

65. Use of the APP RNAi reagent according to any one of claims 1-57 in the manufacture of a pharmaceutical preparation for treating APP-related neurological disorders.

66. The use according to claim 65, wherein the APP-related neurological disease is selected from Alzheimer's disease, Down syndrome, or cerebral amyloid angiopathy.

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