Patent Application
20250064958
The present application is being filed along with a Sequence Listing in ST.26 XML format. The Sequence Listing is provided as a file titled “30578 WO” created Jun. 14, 2024, and is 1.576 megabytes in size. The Sequence Listing information in the ST.26 XML format is incorporated herein by reference in its entirety.
Amyloid precursor protein (APP) is a transmembrane protein expressed in neurons and glia. APP is cleaved by β-secretase and γ-secretase to release the amyloid beta (Aβ) peptides, which encompass a group of peptides ranging in size of 38-43 amino acid residues. Aβ monomers aggregate into various types of higher order structures including oligomers, protofibrils and amyloid fibrils. Amyloid oligomers are soluble and may spread throughout the brain, while amyloid fibrils are larger and insoluble and can further aggregate to form amyloid deposits or plaques. Amyloid plaques in the brain have been associated with a number of conditions and diseases, including Alzheimer's disease (AD), Down's syndrome, and cerebral amyloid angiopathy (CAA).
The blood brain barrier (BBB) is a selective semipermeable border of capillary endothelial cells that prevents solutes, including pathogens, from passing into the central nervous system (CNS). The BBB allows the passage of some small molecules by passive diffusion and the cells of BBB actively transport metabolic products crucial to neural function such as glucose and amino acids across the barrier using specific transport proteins. The BBB has neuroprotective function by tightly controlling access to the brain; but it also impedes access of therapeutic agents to CNS. Antibodies directed to transferrin receptor (“TfR”) have been used for modulating BBB transport. However, attempts at using anti-TfR antibodies to shuttle therapeutic agents across the BBB have proven challenging. To date, there are no approved TfR shuttles or conjugates for the treatment of CNS diseases.
RNA interference (RNAi) is a highly conserved regulatory mechanism in which RNA molecules are involved in sequence-specific suppression of gene expression by double-stranded RNA molecules (dsRNA) (Fire et al., Nature 391:806-811, 1998).
Currently, there are no disease modifying treatments available for Down's syndrome and cerebral amyloid angiopathy. Although FDA recently approved two anti-Aβ antibodies (aducanumab and lecanemab) for treating AD, the AD patients vary widely in the progression of disease, initiation of symptoms, trajectory of cognitive and functional decline, and their response to treatment. Accordingly, there remains a need for therapeutic agents that can cross BBB and access the CNS and attack the initiation of amyloid cascade by inhibiting APP mRNA expression, e.g., by utilizing RNAi, and thereby reduce the production and/or level of disease causing Aβ peptides.
Provided herein are APP RNAi agents and compositions comprising an APP RNAi agent that can access CNS and reduce APP mRNA expression. Also provided herein are methods of using the APP RNAi agents or compositions comprising an APP RNAi agent for reducing APP expression and/or treating APP associated neurological diseases.
In one aspect, provided herein are APP RNAi agents comprising Formula (I): (R-L)n-P, wherein R is a double stranded RNA (dsRNA) comprising a sense stand and an antisense strand, and the antisense strand is complementary to APP mRNA; wherein P is a protein comprising one monovalent human TfR binding domain (“human TfR binding protein”); wherein L is a linker, or optionally absent, and wherein n is an integer of 1 to 3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3.
In some embodiments, provided herein are APP RNAi agents comprising Formula (I): (R-L)n-P, wherein R is a double stranded RNA (dsRNA) comprising a sense stand and an antisense strand, and the antisense strand is complementary to APP mRNA; wherein P is a protein comprising one monovalent human TfR binding domain; wherein L is a linker, or optionally absent, wherein 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 wherein n is an integer of 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 to SEQ ID NO: 7 and VL comprises a sequence having at least 95% sequence identity to SEQ ID NO: 8. Exemplary sequences of human TfR binding domains and proteins are provided in Table 1a and 1b.
In some embodiments, L is a Mal-Tet-TCO linker, SMCC linker, or GDM linker (see Table 4). In some embodiments, L is a SMCC linker in Table 4.
In another aspect, provided herein are APP RNAi agents comprising a double stranded RNA (dsRNA) comprising a sense stand and an antisense strand, wherein the sense stand and antisense strand sequences are selected from Table 5a, 5b, 7a, 7b. In some embodiments, APP RNAi agents comprising any dsRNA in Table 5a, 5b, 7a, 7b.
Exemplary unmodified sense strand and antisense strand sequences of dsRNA targeting human APP mRNA are provided in Table 5a and 5b. In some embodiments, the sense strand and the antisense strand of the dsRNA comprise a pair of nucleic acid sequences selected from the group consisting of:
The dsRNA can include modifications. The modifications can be made to one or more nucleotides of the sense and/or antisense strand or to the internucleotide linkages. In some embodiments, one or more nucleotides of the sense strand and/or the antisense strand are independently modified nucleotides, which means the sense strand and the antisense strand can 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′-fluoro modified nucleotide, 2′-O-methyl modified nucleotide, 2′ deoxy nucleotide (DNA), or 2′-O-alkyl modified nucleotide. In some embodiments, each nucleotide of the sense strand and the antisense strand is independently a modified nucleotide, e.g., a 2′-fluoro modified nucleotide, 2′-O-methyl modified nucleotide, 2′ deoxy nucleotide (DNA), or 2′-O-alkyl modified nucleotide.
In some embodiments, the sense strand has four 2′-fluoro modified nucleotides, e.g., at positions 7, 9, 10, 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 2′ deoxy nucleotide (DNA). In some embodiments, the other nucleotides of the sense strand are 2′-O-methyl modified nucleotides. In some embodiments, the antisense strand has four 2′-fluoro modified nucleotides, e.g., at positions 2, 6, 14, 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.
In some embodiments, the sense strand has three 2′-fluoro modified nucleotides, e.g., at positions 9, 10, 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 2′ deoxy nucleotide (DNA). In some embodiments, the other nucleotides of the sense strand are 2′-O-methyl modified nucleotides. In some embodiments, the antisense strand has five 2′-fluoro modified nucleotides, e.g., at positions 2, 5, 7, 14, 16 from the 5′ end of the antisense strand. In some embodiments, the antisense strand has five 2′-fluoro modified nucleotides, e.g., at positions 2, 5, 8, 14, 16 from the 5′ end of the antisense strand. In some embodiments, the antisense strand has five 2′-fluoro modified nucleotides, e.g., at positions 2, 3, 7, 14, 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.
In some embodiments, the 5′ end of the antisense strand has a phosphate analog, e.g., 5′-vinylphosphonate (5′-VP).
In some embodiments, the sense strand or the antisense strand comprises an abasic moiety or inverted abasic moiety.
In some embodiments, the sense strand and the antisense strand have one or more modified internucleotide linkages. In some embodiments, the modified internucleotide linkage is phosphorothioate linkage. In some embodiments, the sense strand has four or five phosphorothioate linkages. In some embodiments, the antisense strand has four or five phosphorothioate linkages. In some embodiments, the sense strand and the antisense strand each has four or five phosphorothioate linkages. In some embodiments, the sense strand has four phosphorothioate linkages and the antisense strand has five phosphorothioate linkages.
Exemplary modified sense strand and antisense strand sequences of dsRNA targeting human APP mRNA are provided in Table 7a and 7b.
In some embodiments, provided herein are APP RNAi agents comprising Formula (I): (R-L)n-P, wherein R is a double stranded RNA (dsRNA) comprising a sense stand and an antisense strand, and the antisense strand is complementary to APP mRNA; wherein P is a protein comprising one 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., a SMCC linker in Table 4).
In some embodiments, provided herein are APP RNAi agents comprising Formula (I): (R-L)n-P, wherein R is a double stranded RNA (dsRNA) comprising a sense stand and an antisense strand, and the dsRNA is any dsRNA in Table 5a, 5b, 7a or 7b (e.g., dsRNA No. 1); wherein P is a protein comprising one monovalent human TfR binding domain; wherein L is a linker, or optionally absent, and wherein n is an integer of 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., a SMCC linker in Table 4).
In some embodiments, provided herein are APP RNAi agents comprising Formula (I): (R-L)n-P, wherein R is a double stranded RNA (dsRNA) comprising a sense stand and an antisense strand, and the dsRNA is any dsRNA in Table 5a, 5b, 7a or 7b (e.g., dsRNA No. 1); wherein P is a protein comprising one 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 of 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., a SMCC linker in Table 4).
In some embodiments, provided herein are APP RNAi agents comprising Formula (I): (R-L)n-P, wherein R is a double stranded RNA (dsRNA) comprising a sense stand and an antisense strand, and the antisense strand is complementary to APP mRNA; wherein P is a protein comprising one 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 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 in Table 4 (e.g., a SMCC linker in Table 4).
In some embodiments, provided herein are APP RNAi agents comprising Formula (I): (R-L)n-P, wherein R is a double stranded RNA (dsRNA) comprising a sense stand and an antisense strand, and the antisense strand is complementary to APP mRNA; wherein P is a protein comprising one 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 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 in Table 4 (e.g., a SMCC linker in Table 4).
In some embodiments, provided herein are APP RNAi agents comprising Formula (I): (R-L)n-P, wherein R is a double stranded RNA (dsRNA) comprising a sense stand 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 one monovalent human TfR binding domain; wherein L is a linker, or optionally absent, and wherein n is an integer of 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., a SMCC linker in Table 4).
In some embodiments, provided herein are APP RNAi agents comprising Formula (I): (R-L)n-P, wherein R is a double stranded RNA (dsRNA) comprising a sense stand 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 one 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 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 in Table 4 (e.g., a SMCC linker in Table 4).
In some embodiments, provided herein are APP RNAi agents comprising Formula (I): (R-L)n-P, wherein R is a double stranded RNA (dsRNA) comprising a sense stand 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 one 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 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 in Table 4 (e.g., a SMCC linker in Table 4).
In some embodiments, provided herein are APP RNAi agents comprising Formula (I): (R-L)n-P, wherein R is a double stranded RNA (dsRNA) comprising a sense stand 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 one monovalent human TfR binding domain; wherein L is a linker, or optionally absent, and wherein n is an integer of 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., a SMCC linker in Table 4).
In some embodiments, provided herein are APP RNAi agents comprising Formula (I): (R-L)n-P, wherein R is a double stranded RNA (dsRNA) comprising a sense stand 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 one 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 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 in Table 4 (e.g., a SMCC linker in Table 4).
In some embodiments, provided herein are APP RNAi agents comprising Formula (I): (R-L)n-P, wherein R is a double stranded RNA (dsRNA) comprising a sense stand 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 one 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 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 in Table 4 (e.g., a SMCC linker in Table 4).
In another aspect, provided herein are methods of treating an APP associated neurologic disease in a patient in need thereof, and such the method comprises administering to the patient an effective amount of the APP RNAi agent or a pharmaceutical composition described herein. In some embodiments, the APP associated neurological disease is selected from Alzheimer's disease, Down's syndrome, or cerebral amyloid angiopathy. The APP RNAi agent or a pharmaceutical composition comprising APP RNAi agent can be administered to the patient intravenously or subcutaneously.
In another aspect, provided herein are APP RNAi agents or pharmaceutical compositions comprising an APP RNAi agent for use in a therapy. Also provided herein are APP RNAi agents or pharmaceutical compositions comprising an APP RNAi agent for use in the treatment of an APP associated neurological disease, e.g., Alzheimer's disease, Down's syndrome, or cerebral amyloid angiopathy. Also provided herein are uses of the APP RNAi agent in the manufacture of a medicament for treating an APP associated neurological disease, e.g., Alzheimer's disease, Down's syndrome, or cerebral amyloid angiopathy.
Provided herein are APP RNAi agents and compositions comprising an APP RNAi agent that can access CNS and reduce APP mRNA expression. Also provided herein are methods of using the APP RNAi agents or compositions comprising an APP RNAi agent for reducing APP expression and/or treating APP associated neurological diseases.
In one aspect, provided herein are APP RNAi agents comprising Formula (I): (R-L)n-P, wherein R is a double stranded RNA (dsRNA) comprising a sense stand and an antisense strand, wherein the antisense strand is complementary to APP mRNA; wherein P is a protein comprising one monovalent human TfR binding domain (“human TfR binding protein”); wherein L is a linker, or optionally absent, and wherein n is an integer of 1 to 3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3.
In some embodiments, provided herein are APP RNAi agents comprising Formula (I): (R-L)n-P, wherein R is a double stranded RNA (dsRNA) comprising a sense stand and an antisense strand, and the antisense strand is complementary to APP mRNA; wherein P is a protein comprising one monovalent human TfR binding domain; wherein L is a linker, or optionally absent, wherein 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 wherein n is an integer of 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., a SMCC linker in Table 4).
In some embodiments, provided herein are APP RNAi agents comprising Formula (I): (R-L)n-P, wherein R is a double stranded RNA (dsRNA) comprising a sense stand and an antisense strand, and the antisense strand is complementary to APP mRNA; wherein P is a protein comprising one 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., a SMCC linker in Table 4).
In some embodiments, provided herein are APP RNAi agents comprising Formula (I): (R-L)n-P, wherein R is a double stranded RNA (dsRNA) comprising a sense stand and an antisense strand, and the dsRNA is any dsRNA in Table 5a, 5b, 7a or 7b (e.g., dsRNA No. 1); wherein P is a protein comprising one monovalent human TfR binding domain; wherein L is a linker, or optionally absent, and wherein n is an integer of 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., a SMCC linker in Table 4).
In some embodiments, provided herein are APP RNAi agents comprising Formula (I): (R-L)n-P, wherein R is a double stranded RNA (dsRNA) comprising a sense stand and an antisense strand, and the dsRNA is any dsRNA in Table 5a, 5b, 7a or 7b (e.g., dsRNA No. 1); wherein P is a protein comprising one 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 of 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., a SMCC linker in Table 4).
In another aspect, provided herein are APP RNAi agents comprising a double stranded RNA (dsRNA) comprising a sense stand and an antisense strand, wherein the sense stand and antisense strand sequences are selected from Table 5a, 5b, 7a, 7b. In some embodiments, APP RNAi agents comprising any dsRNA in Table 5a, 5b, 7a, 7b.
The APP RNAi agents described herein comprise a protein comprising one monovalent human TfR binding domain (“human TfR binding protein”). Human TfR binding protein of the APP RNAi agents can bind TfR on BBB and transport the dsRNA into the CNS.
Exemplary sequences of human TfR binding domains and proteins are provided in Table 1a and 1b. In some embodiments, the monovalent human TfR binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), and 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 TD NO: 1, HCDR2 comprises SEQ TD NO: 2, HCDR3 comprises SEQ TD NO: 3, LCDR1 comprises SEQ ID NO: 4, LCDR2 comprises SEQ TD NO: 5, and LCDR3 comprises SEQ ID NO: 6. In some embodiments, VH comprises SEQ TD NO: 7, and VL comprises SEQ TD NO: 8. In some embodiments, VH comprises a sequence having at least 9500 sequence identity to SEQ ID NO: 7, and VL comprises a sequence having at least 95% sequence identity to SEQ TD NO: 8.
In some embodiments, the monovalent human TfR binding domain is an antibody fragment, e.g., 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.
In some embodiments, the human TfR binding protein further comprises a half-life extender, e.g., an immunoglobulin Fc region or a VHH that binds human serum albumin (HSA).
In some embodiments, the human TfR binding protein further comprises an immunoglobulin Fc region, e.g., 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 comprising proline at residue 228, and alanine at residues 234 and 235 (all residues are numbered according to the EU Index numbering, also called hIgG4PAA Fc region). In some embodiments, the human TfR binding protein further comprises a modified human IgG1 Fc region comprising alanine at residues 234, 235, and 329, serine at position 265, aspartic acid at position 436 (all residues are numbered according to the EU Index numbering, also called hIgG1 effector null or hIgG1EN Fc region).
In some embodiments, the human TfR binding protein further comprise a VHH that binds human HSA. In some embodiments, the VHH also binds mouse, rat, and/or Cynomolgus monkey albumin. An exemplary VHH that binds human HSA is shown in Table 2. In some embodiments, such a VHH comprises CDR1 comprising SEQ ID NO: 20, CDR2 comprising SEQ ID NO: 21, and CDR3 comprising SEQ ID NO: 22. In some embodiments, such a VHH comprises SEQ ID NO: 23. In some embodiments, the VHH is linked to the TfR binding domain through a peptide linker, e.g., (GGGGQ)4 (SEQ ID NO: 24). In some embodiments, the VHH is linked to the C-terminus of the TfR binding domain.
In some embodiments, the human TfR binding protein is heterodimeric antibody that comprises a first arm comprising one monovalent human TfR binding domain and a second arm that is a null arm, e.g., an arm that does not bind any known human target (e.g., an isotype arm). Heterodimeric antibodies such as heteromab, orthomab or duobody have been described in WO2014150973, WO2016118742, WO2018118616, and WO2011131746. In some embodiments, the first arm comprises any monovalent human TfR binding domain described herein. In some embodiments, the second arm is a null arm that does not bind any known human target (e.g., an isotype arm) comprises the sequences in Table 1a. In some embodiments, the second arm comprises a heavy chain (HC) and a light chain (LC), wherein the HC comprises SEQ ID NO: 18, and the LC comprises SEQ ID NO: 19.
In some embodiments, the human TfR binding protein comprises heterodimeric mutations. In some embodiments, the human TfR binding protein comprises a modified Fc region comprising a first Fc CH3 domain comprising serine at residue 349, methionine at residue 366, tyrosine at residue 370, and valine at residue 409, and a second Fc CH3 domain comprising glycine at residue 356, aspartic acid at residue 357, glutamine at residue 364 and alanine at residue 407 (all residues are numbered according to the EU Index numbering). In some embodiments, the human TfR binding protein comprises a modified Fc region comprising a first Fc CH3 domain comprising leucine at residue 405, and a second Fc CH3 domain comprising arginine at residue 409 (all residues are numbered according to the EU Index numbering).
In some embodiments, the human TfR binding protein comprises one or more native cysteine residues, which can be used for conjugation. For example, in some embodiments, the human TfR binding protein comprises a native cysteine at position 220 of the light chain and/or a native cysteine at position 226 of the heavy chain, which can be used for conjugation (all residues according to the EU Index numbering).
In some embodiments, the human TfR binding protein comprises engineered cysteine residues for conjugation. The approach of including engineered cysteines as a means for conjugation has been described in WO 2018/232088. In some embodiments, the human TfR binding protein comprises a heavy chain comprising one or more cysteines at the following residues: 124, 157, 162, 262, 373, 375, 378, 397, 415 (all residues according to the EU Index numbering). In some embodiments, the human TfR binding protein comprises a light chain (e.g., a kappa light chain) comprising one or more cysteines at the following residues: 156, 171, 191, 193, 202, 208 (all residues according to the EU Index numbering). In some embodiments, the human TfR binding protein comprises a heavy chain constant region comprising cysteine at residue 124 (according to the EU Index numbering). In some embodiments, the human TfR binding protein comprises a light chain constant region comprising cysteine at residue 156 (according to the EU Index numbering). In some embodiments, the human TfR binding protein comprises an immunoglobulin Fc region comprising cysteine at residue 378 (according to the EU Index numbering).
In some embodiments, the human TfR binding protein is any one of the human TfR binding proteins in Table 1b, e.g., TBP1, TBP2, TBP3, TBP4, TBP5.
In some embodiments, the human TfR binding protein has a Fab format, e.g., TBP1. In some embodiments, the human TfR binding protein comprises one HC and one LC, and wherein the HC comprises SEQ ID NO: 9 and the LC comprises SEQ ID NO: 10.
In some embodiments, the human TfR binding protein has a Fab-VHH format, e.g., TBP2. In some embodiments, the human TfR binding proteins comprises one HC and one LC, wherein the HC comprises SEQ ID NO: 11 and the LC comprises SEQ ID NO: 12 or 10.
In some embodiments, the human TfR binding protein has a heterodimeric antibody format, e.g., 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.
In some embodiments, the human TfR binding protein has a one arm heteromab format, e.g., 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, HC2 comprises SEQ ID NO: 15. In some embodiments, provided herein are human TfR binding proteins comprise 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.
The human TfR binding proteins described herein can be recombinantly produced in a host cell, for example, using an expression vector. For example, an expression vector may include a sequence that encodes one or more signal peptides that facilitate secretion of the polypeptide(s) from a host cell. Expression vectors containing a polynucleotide of interest (e.g., a polynucleotide encoding a heavy chain or light chain of the TfR binding proteins) may be transferred into a host cell by well-known methods. Additionally, expression vectors may contain one or more selection markers, e.g., tetracycline, neomycin, and dihydrofolate reductase, to aide in detection of host cells transformed with the desired polynucleotide sequences.
A host cell includes cells stably or transiently transfected, transformed, transduced or infected with one or more expression vectors expressing all or a portion of the TfR binding proteins described herein. According to some embodiments, a host cell may be stably or transiently transfected, transformed, transduced or infected with an expression vector expressing HC polypeptides and an expression vector expressing LC polypeptides of the TfR binding proteins described herein. In some embodiments, a host cell may be stably or transiently transfected, transformed, transduced or infected with an expression vector expressing HC and LC polypeptides of the TfR binding proteins described herein. The TfR binding proteins may be produced in mammalian cells such as CHO, NS0, HEK293 or COS cells according to techniques well known in the art.
Medium, into which the TfR binding proteins has been secreted, may be purified by conventional techniques, such as mixed-mode methods of ion-exchange and hydrophobic interaction chromatography. For example, the medium may be applied to and eluted from a Protein A or G column using conventional methods; mixed-mode methods of ion-exchange and hydrophobic interaction chromatography may also be used. Soluble aggregate and multimers may be effectively removed by common techniques, including size exclusion, hydrophobic interaction, ion exchange, or hydroxyapatite chromatography. Various methods of protein purification may be employed, and such methods are known in the art and described, for example, in Deutscher, Methods in Enzymology 182: 83-89 (1990) and Scopes, Protein Purification: Principles and Practice, 3rd Edition, Springer, NY (1994).
Some APP RNAi agents used in the Examples below comprise a protein comprising one monovalent mouse TfR binding domain (“mouse TfR binding proteins” or mTBP). Exemplary sequences of mouse TfR binding proteins are provided in Table 3. Such APP RNAi agents comprising a mouse TfR binding protein can serve as surrogate molecules in mouse models for APP RNAi agents comprising a human TfR binding protein.
In some embodiments, the APP RNAi agents described herein comprises a linker that links the human TfR binding protein to the dsRNA. In some embodiments, the linker is a Mal-Tet-TCO linker, SMCC linker, or GDM linker (structures of these linkers shown in Table 4). In some embodiments, the linker is a SMCC linker.
dsRNA
The APP RNAi agents described herein comprise a double stranded RNA (dsRNA) comprising a sense stand 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), the RISC can bind and degrade target APP mRNA.
In some embodiments, the sense strand and the antisense strand of the dsRNA are each 15-30 nucleotides in length, e.g., 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 strand and antisense strand of the dsRNA may have overhangs at either the 5′ end or the 3′ end (i.e., 5′ overhang or 3′ overhang). For example, the sense strand and the antisense strand may have 5′ or 3′ overhangs of 1 to 5 nucleotides or 1 to 3 nucleotides. In some embodiments, the antisense strand comprises a 3′ overhang of two nucleotides.
Exemplary unmodified sense strand and antisense strand sequences of dsRNA targeting human APP mRNA are provided in Table 5a and 5b.
Table 5b. Unmodified Nucleic Acid Sequences of dsRNA Targeting the Coding Sequence of Human APP mRNA
In some embodiments, the sense strand and the antisense strand of the dsRNA comprise a pair of nucleic acid sequences selected from the group consisting of:
The dsRNA can include modifications. The modifications can be made to one or more nucleotides of the sense and/or antisense strand or to the internucleotide linkages, which are the bonds between two nucleotides in the sense or antisense strand. For example, some 2′-modifications of ribose or deoxyribose can increase RNA or DNA stability and half-life. Such 2′-modifications can be 2′-fluoro, 2′-O-methyl (i.e., 2′-methoxy), or 2′-O-alkyl.
In some embodiments, one or more nucleotides of the sense strand and/or the antisense strand are independently modified nucleotides, which means the sense strand and the 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′-fluoro modified nucleotide, 2′-O-methyl modified nucleotide, 2′ deoxy nucleotide (DNA), or 2′-O-alkyl modified nucleotide. In some embodiments, each nucleotide of the sense strand and the antisense strand is independently a modified nucleotide, e.g., a 2′-fluoro modified nucleotide, 2′-O-methyl modified nucleotide, 2′ deoxy nucleotide (DNA), or 2′-O-alkyl modified nucleotide. In some embodiments, at least one nucleotide of the sense strand is 2′ deoxy nucleotide (DNA).
In some embodiments, the sense strand has four 2′-fluoro modified nucleotides, e.g., at positions 7, 9, 10, 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 2′ deoxy nucleotide (DNA). In some embodiments, the other nucleotides of the sense strand are 2′-O-methyl modified nucleotides.
In some embodiments, the antisense strand has four 2′-fluoro modified nucleotides, e.g., at positions 2, 6, 14, 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.
In some embodiments, the sense strand has three 2′-fluoro modified nucleotides, e.g., at positions 9, 10, 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 2′ deoxy nucleotide (DNA). In some embodiments, the other nucleotides of the sense strand are 2′-O-methyl modified nucleotides.
In some embodiments, the antisense strand has five 2′-fluoro modified nucleotides, e.g., at positions 2, 5, 7, 14, 16 from the 5′ end of the antisense strand. In some embodiments, the antisense strand has five 2′-fluoro modified nucleotides, e.g., at positions 2, 5, 8, 14, 16 from the 5′ end of the antisense strand. In some embodiments, the antisense strand has five 2′-fluoro modified nucleotides, e.g., at positions 2, 3, 7, 14, 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.
In some embodiments, the 5′ end of the antisense strand has a phosphate analog, e.g., 5′-vinylphosphonate (5′-VP).
In some embodiments, the sense strand or the antisense strand comprises an abasic moiety or inverted abasic moiety, e.g., a moiety shown in Table 6. In some embodiments, the inverted basic moiety or abasic moiety increases stability of the sense strand or the antisense strand. In some embodiments, the sense strand comprises an inverted abasic moiety. In some embodiments, the antisense strand comprises an inverted abasic moiety.
In some embodiments, the sense strand and the antisense strand have one or more modified internucleotide linkages. In some embodiments, the modified internucleotide linkage is phosphorothioate linkage. In some embodiments, the sense strand has four or five phosphorothioate linkages. In some embodiments, the antisense strand has four or five phosphorothioate linkages. In some embodiments, the sense strand and the antisense strand each has four or five phosphorothioate linkages. In some embodiments, the sense strand has four phosphorothioate linkages and the antisense strand has five phosphorothioate linkages.
Exemplary modified sense strand and antisense strand sequences of dsRNA targeting human APP mRNA are provided in Table 7a and 7b.
In some embodiments, the dsRNA comprises a sense strand that comprises a sequence that has 1, 2, or 3 differences from a sense stand sequence in Table 7a or 7b. In some embodiments, the dsRNA comprises an antisense strand that comprises a sequence that has 1, 2, or 3 differences from an antisense stand sequence in Table 7a or 7b.
In some embodiments, the sense strand and the antisense strand of the dsRNA comprise a pair of nucleic acid sequences selected from the group consisting of:
In some embodiments, the sense strand and the antisense strand of the dsRNA have a pair of nucleic acid sequences selected from the group consisting of:
In some embodiments, the sense strand comprises SEQ ID NO: 172, and the antisense strand comprises SEQ ID NO: 217. In some embodiments, the sense strand consists of SEQ ID NO: 172, and the antisense strand consists of SEQ ID NO: 217.
The sense strand and antisense strand of dsRNA can be synthesized using any nucleic acid polymerization methods known in the art, for example, solid-phase synthesis by employing phosphoramidite chemistry methodology (e.g., Current Protocols in Nucleic Acid Chemistry, Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA), H-phosphonate, phosphortriester chemistry, or enzymatic synthesis. Automated commercial synthesizers can be used, for example, MerMade™ 12 from LGC Biosearch Technologies, or other synthesizers from BioAutomation or Applied Biosystems. Phosphorothioate linkages can be introduced using a sulfurizing reagent such as phenylacetyl disulfide or DDTT (((dimethylaminomethylidene) amino)-3H-1,2,4-dithiazaoline-3-thione). It is well known to use similar techniques and commercially available modified amidites and controlled-pore glass (CPG) products to synthesize modified oligonucleotides or conjugated oligonucleotides.
Purification methods can be used to exclude the unwanted impurities from the final oligonucleotide product. Commonly used purification techniques for single stranded oligonucleotides include reverse-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, oligonucleotides can be analyzed by mass spectrometry and quantified by spectrophotometry at a wavelength of 260 nm. The sense strand and antisense strand can then be annealed to form a dsRNA.
The RNAi agent described herein can be made by a variety of procedures known to one of ordinary skill in the art, some of which are illustrated in the preparations and examples below, e.g., in Examples 1-3. One of ordinary skill in the art recognizes that the specific synthetic steps for each of the routes described may be combined in different ways, or in conjunction with steps from different schemes, to prepare the RNAi agent. The product of each step can be recovered by conventional methods well known in the art, including extraction, evaporation, precipitation, chromatography, filtration, trituration, and crystallization. The reagents and starting materials are readily available to one of ordinary skill in the art.
In some embodiments, the TfR binding protein with native or engineered cysteines described herein can be first treated with a reducing agent, e.g., DTT, and then re-oxidized with an oxidizing agent, e.g., DHAA. The resulting oxidized TfR binding protein is then incubated with a linker functionalized dsRNA, e.g., linker-dsRNA, to produce the conjugated RNAi agent.
In another aspect, provided herein are pharmaceutical compositions comprising any of the APP RNAi agents described herein and a pharmaceutically acceptable carrier. Such pharmaceutical compositions can also comprise one or more pharmaceutically acceptable excipient, diluent, or carrier. Pharmaceutical compositions can be prepared by methods well known in the art (e.g., Remington: The Science and Practice of Pharmacy, 23rd edition (2020), A. Loyd et al., Academic Press).
In another aspect, provided herein are methods of treating an APP associated neurologic disease in a patient in need thereof, and such the method comprises administering to the patient an effective amount of the APP RNAi agent or a pharmaceutical composition described herein. In some embodiments, the APP associated neurological disease is selected from Alzheimer's disease, Down's syndrome, or cerebral amyloid angiopathy. The APP RNAi agent or a pharmaceutical composition comprising APP RNAi agent can be administered to the patient intravenously or subcutaneously.
APP RNAi agent dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.
Dosage values may vary with the type and severity of the condition to be alleviated. It is further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions.
In another aspect, provided herein are APP RNAi agents or pharmaceutical compositions comprising an APP RNAi agent for use in a therapy. Also provided herein are APP RNAi agents or pharmaceutical compositions comprising an APP RNAi agent for use in the treatment of an APP associated neurological disease, e.g., Alzheimer's disease, Down's syndrome, or cerebral amyloid angiopathy. Also provided herein are uses of the APP RNAi agent in the manufacture of a medicament for treating an APP associated neurological disease, e.g., Alzheimer's disease, Down's syndrome, or cerebral amyloid angiopathy.
As used herein, the terms “a,” “an,” “the,” and similar terms used in the context of the present disclosure (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context.
The term “about” as used herein, means in reasonable vicinity of the stated numerical value, such as plus or minus 10% of the stated numerical value.
As used herein, the term “alkyl” means saturated linear or branched-chain monovalent hydrocarbon radical, containing the indicated number of carbon atoms. For example, “C1-C20 alkyl” means a radical having 1-20 carbon atoms in a linear or branched arrangement.
The term “antibody,” as used herein, refers to a molecule that binds an antigen. Embodiments of an antibody include a monoclonal antibody, polyclonal antibody, human antibody, humanized antibody, chimeric antibody, heterodimeric antibody, bispecific or multispecific antibody, or conjugated antibody. The antibodies can be of any class (e.g., IgG, IgE, IgM, IgD, IgA), and any subclass (e.g., IgG1, IgG2, IgG3, IgG4).
An immunoglobulin G (IgG) type antibody comprised of four polypeptide chains: two heavy chains (HC) and two light chains (LC) that are cross-linked via inter-chain disulfide bonds. The amino-terminal portion of each of the four polypeptide chains includes a variable region of about 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 is comprised of a heavy chain variable region (VH) and a heavy chain constant region. Each light chain is comprised of a light chain variable region (VL) and a light chain constant region. The IgG isotype may be further divided into subclasses (e.g., IgG1, IgG2, IgG3, and IgG4).
The VH and VL regions can be further subdivided into regions of hyper-variability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). The CDRs are exposed on the surface of the protein and are important regions of the antibody for antigen binding specificity. Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Herein, 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”. The CDRs contain most of the residues that form specific interactions with the antigen. Assignment of amino acid residues to the CDRs may be done according to the well-known schemes, including those described in 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 available on at www.imgt.org; see Lefranc et al., Nucleic Acids Res. 1999; 27:209-212).
Embodiments of the present disclosure also include antibody fragments or antigen-binding fragments that, as used herein, comprise at least a portion of an antibody retaining the ability to specifically interact with an antigen or an epitope of the antigen, such as Fab, Fab′, F(ab′)2, Fv fragments, scFv antibody fragments, scFab, disulfide-linked Fvs (sdFv), a Fd fragment.
The term “antigen binding domain”, as used herein, refers to a portion of an antibody or antibody fragment that binds an antigen or an epitope of the antigen. For example. “TfR binding domain” refers to a portion of an antibody or antibody fragment that binds TfR or an epitope of TfR.
The term “heterodimeric antibody”, as used herein, refers to an antibody that comprises two distinct antigen-binding domains.
As used herein, “antisense strand” means a single-stranded oligonucleotide that is complementary to a region of a target sequence. Likewise, and as used herein, “sense strand” means a single-stranded oligonucleotide that is complementary to a region of an antisense strand.
As used herein, “APP” (also known as amyloid beta precursor protein or ABPP) refers to an amyloid precursor protein (APP) mRNA transcript, protein, or polypeptide. The nucleotide sequences of human APP mRNA transcript variants and amino acid sequences of human APP protein isoforms can be found at:
The human APP mRNA transcript variant 1 sequence encoding human APP protein isoform a (longest isoform) can be found at NM_000484.4:
The nucleic acid sequence of a mouse APP mRNA transcript can be found at NM_001198823.1; and the amino acid sequence of a mouse APP protein can be found at NP_001185752.1. The nucleic acid sequence of a rat APP mRNA transcript can be found at NM_019288.2; and the amino acid sequence of a rat APP protein can be found at NP_062161.1. The nucleic acid sequence of a monkey APP mRNA transcript can be found at XM_015133068.2; and the amino acid sequence of a monkey APP protein can be found at XP_014988554.1.
As used herein, the term “APP associated neurological disease” refers to a neurological disease characterized by extracellular amyloid deposits or plaques.
The terms “bind” and “binds” as used herein are intended to mean, unless indicated otherwise, the ability of a protein or molecule to form a chemical bond or attractive interaction with another protein or molecule, which results in proximity of the two proteins or molecules as determined by common methods known in the art.
As used herein, “complementary” means a structural relationship between two nucleotides (e.g., on two opposing nucleic acids or on opposing regions of a single nucleic acid strand, e.g., a hairpin) that permits the two nucleotides to form base pairs with one another. For example, a purine nucleotide of one nucleic acid that is complementary to a pyrimidine nucleotide of an opposing nucleic acid may base pair together by forming hydrogen bonds with one another. Complementary nucleotides can base pair in the Watson-Crick manner or in any other manner that allows for the formation of stable duplexes. Likewise, two nucleic acids may have regions of multiple nucleotides that are complementary with each other to form regions of complementarity, as described herein.
As used herein, “duplex,” in reference to nucleic acids or oligonucleotides, means a structure formed through complementary base pairing of two antiparallel sequences of nucleotides (i.e., in opposite directions), whether formed by two separate nucleic acid strands or by a single, folded strand (e.g., via a hairpin).
An “effective amount” refers to an amount necessary (for periods of time and for the means of administration) to achieve the desired therapeutic result. An effective amount of a protein or conjugate may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the protein or conjugate to elicit a desired response in the individual. An effective amount is also one in which any toxic or detrimental effects of the protein or conjugate are outweighed by the therapeutically beneficial effects.
The term “Fc region” as used herein refers to a polypeptide comprising the CH2 and CH3 domains of a constant region of an immunoglobulin, e.g., IgG1, IgG2, IgG3, or IgG4. Optionally, the Fc region may include a portion of the hinge region 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, e.g., a 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 with reduced or eliminated effector functions compared to the corresponding wild type IgG Fc region. The numbering of the residues 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: U.S. Dept. of Health and Human Services, Public Health Service, National Institutes of Health, 1991). The boundaries of the Fc region of an immunoglobulin heavy chain might vary, and the human IgG heavy chain Fc region is usually defined as the stretch from the N-terminus of the CH2 domain (e.g., the amino acid residue at position 231 according to the EU index numbering) to the C-terminus of the CH3 domain (or the C-terminus of the immunoglobulin).
The term “knockdown” or “expression knockdown” refers to reduced mRNA or protein expression of a gene or target after treatment of a reagent.
As used herein, “modified internucleotide linkage” means an internucleotide linkage having one or more chemical modifications when compared with a reference internucleotide linkage having a phosphodiester bond. A modified internucleotide linkage can be a non-naturally occurring linkage. In some embodiments, the modified internucleotide linkage is phosphorothioate linkage.
As used herein, “modified nucleotide” refers to a nucleotide having one or more chemical modifications when compared with a 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 can have, for example, one or more chemical modification in its sugar, nucleobase, and/or phosphate group. Additionally, or alternatively, a modified nucleotide can have one or more chemical moieties conjugated to a corresponding reference nucleotide. In some embodiments, the modified nucleotide is a 2′-fluoro modified nucleotide, 2′-O-methyl modified nucleotide, 2′ deoxy nucleotide (DNA), or 2′-O-alkyl modified nucleotide. In some embodiments, the modified nucleotide has a phosphate analog, e.g., 5′-vinylphosphonate. In some embodiments, the modified nucleotide has an abasic moiety or inverted abasic moiety, e.g., a moiety shown in Table 6.
As used herein, “nucleotide” means an organic compound having a nucleoside (a nucleobase, e.g., adenine, cytosine, guanine, thymine, or uracil, and a pentose sugar, e.g., ribose or 2′-deoxyribose) linked to a phosphate group. A “nucleotide” can serve as a monomeric unit of nucleic acid polymers such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
As used herein, a “null arm” means an antibody arm that does not bind any known human target.
As used herein, “oligonucleotide” means a polymer of linked nucleotides, each of which can be modified or unmodified. An oligonucleotide is typically less than about 100 nucleotides in length.
As used herein, “overhang” means the unpaired nucleotide or nucleotides that protrude from the duplex structure of a double stranded oligonucleotide. An overhang may include one or more unpaired nucleotides extending from a duplex region at the 5′ terminus or 3′ terminus of a double stranded oligonucleotide. The overhang can be a 3′ or 5′ overhang on the antisense strand or sense strand of a double stranded oligonucleotide.
The term “patient”, as used herein, refers to a human patient.
As used herein, “phosphate analog” means a chemical moiety that mimics the electrostatic and/or steric properties of a phosphate group. In some embodiments, a phosphate analog is positioned at the 5′ end of an oligonucleotide in place of a 5′-phosphate, which is sometimes susceptible to enzymatic removal. A 5′ phosphate analog can include a phosphatase-resistant linkage. Examples of phosphate analogs include 5′ methylene phosphonate (5′-MP) and 5′-(E)-vinylphosphonate (5′-VP). In some embodiments, the phosphate analog is 5′-VP.
The term “% sequence identity” or “percentage sequence identity” with respect to a reference nucleic acid sequence is defined as the percentage of nucleotides, nucleosides, or nucleobases in a candidate sequence that are identical with the nucleotides, nucleosides, or nucleobases in the reference nucleic acid sequence, after optimally aligning the sequences and introducing gaps or overhangs, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software programs, for example, those described in Current Protocols in Molecular Biology (Ausubel et al., eds., 1987, Supp. 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 needed to achieve maximal alignment over the full length of the sequences being compared. Percentage of “sequence identity” can be determined by comparing two optimally aligned sequences over a comparison window, where the fragment of the nucleic acid sequence in the comparison window may comprise additions or deletions (e.g., gaps or overhangs) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage can be calculated by determining the number of positions at which the identical nucleotide, nucleoside, or nucleobase occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity. The output is the percent identity of the subject sequence with respect to the query sequence.
The term “polypeptide” or “protein”, as used herein, refers to a polymer of amino acid residues. The term applies to polymers comprising naturally occurring amino acids and polymers comprising one or more non-naturally occurring amino acids.
As used herein, “RNAi,” “RNAi agent,” “iRNA,” “iRNA agent,” or “RNA interference agent” means an agent that mediates sequence-specific degradation of a target mRNA by RNA interference, e.g., via RNA-induced silencing complex (RISC) pathway. In some embodiments, the RNAi agent has a sense strand and an antisense strand, and the sense strand and the antisense strand form a duplex (e.g., a double stranded RNA).
As used herein, “strand” refers to a single, contiguous sequence of nucleotides linked together through internucleotide linkages (e.g., phosphodiester linkages or phosphorothioate linkages). A strand can have two free ends (e.g., a 5′ end and a 3′ end).
As used herein, “treatment” or “treating” refers to all processes wherein there may be a slowing, controlling, delaying, or stopping of the progression of the disorders or disease disclosed herein, or ameliorating disorder or disease symptoms, but does not necessarily indicate a total elimination of all disorder or disease symptoms. Treatment includes administration of a protein or nucleic acid or vector or composition for treatment of a disease or condition in a patient, particularly in a human.
The following examples are offered to illustrate, but not to limit, the claimed inventions.
Antibody against human TfR was generated by immunizing AlivaMab® transgenic mice with the extracellular domains of human Transferrin Receptor 1 protein with a His tag (hR-ECD-6His, SEQ TD NO: 170, see Table 8) and mouse Transferrin Receptor protein (mTfR, SEQ ID NO: 169). Antigen positive B-cells were sorted from pooled spleens. Binding of individual antibodies cloned from those B-cells to his-tagged hTfR-ECD was verified.
Additional antibody against human TfR was generated by immunizing AlivaMab® transgenic mice with the apical domain of human Transferrin Receptor 1 protein with a His tag (hTfR-ApD-6His, SEQ TD NO: 171, see Table 8). Antigen positive B-cells were sorted from pooled spleens. Binding of individual antibodies cloned from those B-cells to his-tagged hTfR-ECD was verified.
Affinity variants of the generated human TfR antibodies were made by systematically introducing mutations into individual CDR of each antibody and the resulting variants were subjected to multiple rounds of selection with decreasing concentrations of antigen and/or increasing periods of dissociation to isolate clones with improved affinities. The sequences of individual variants were used to construct a combinatorial library which was subjected to an additional round of selection with increased stringency to identify additive or synergistic mutational pairings between the individual CDR regions. Individual combinatorial clones are sequenced. The heavy chain and light chain CDRs and VH/VL sequences of the human TfR binding domains and proteins are provided in Table 1a.
Human TfR binding proteins were generated by recombinant DNA technology. Such human TfR binding proteins can be expressed in a mammalian cell line such as HEK293 or CHO, either transiently or stably transfected with an expression system using an optimal predetermined HC:LC vector ratio or a single vector system encoding both HC and LC. Clarified media, into which the protein has been secreted, can be purified using the commonly used techniques.
Binding affinity and binding stoichiometry of the exemplified human TfR binding proteins to human and Cynomolgus TfR was characterized using a surface plasmon resonance assay on a Biacore 8K instrument primed with HBS-EP+ (10 mM Hepes pH7.4+150 mM NaCl+3 mM EDTA+0.05% (w/v) surfactant P20) running buffer and analysis temperature set at 37° C. Target human and Cynomolgous TfR ECDs were immobilized on a CM4 chip (Cytiva P/N 29104989) using standard NHS-EDC amine coupling. The TfR binding proteins were prepared at a final concentration of 0.3, 0.1, 0.033, 0.01, 0.0033, 0.001, 0.00033, 0.0001 μM respectively by dilution of stock solution into running buffer.
Binding analysis was performed in a multi-cycle kinetics manner. Each analysis cycle consists of (1) injection of the lowest to highest concentration proteins over all Fc at 50 μL/min for 140 seconds followed by return to buffer flow for 400 seconds to monitor dissociation phase; (2) regeneration of chip surfaces with injection of 3M magnesium chloride, for 30 seconds at 100 μL/min over all cells; and (3) equilibration of chip surfaces with a 50 μL (30-sec) injection of HBS-EP+. Data were processed using standard double-referencing and fit to a 2-state binding model using Biacore 8K Evaluation software, to determine the association rate (kon, M−1s−1 units), dissociation rate (koff, s−1 units), and Rmax (RU units). The equilibrium dissociation constant (KD) is calculated from the relationship KD=koff/kon, and is in molar units. Results are provided in Table 9.
Single strands (sense and antisense) of the dsRNA duplexes were typically synthesized on solid support via a MerMade™ 12 (LGC Biosearch Technologies) or a similar automated oligonucleotide synthesizer. The sequences of the sense and antisense strands were shown in Table 5a or 5b. The sense strands were synthesized using an appropriate CPG such as 3′-Cholesterol-TEG CNA CPG 500 (LGC Biosearch Technologies) or phthalamido amino C6 lcaa CPG 500 Å (Chemgenes) whereas the antisense strands used standard support (LGC Biosearch Technologies). The oligonucleotides were synthesized via phosphoramidite chemistry at an appropriate scale for in-vitro or in-vivo experimentation.
Standard reagents were used in the oligo synthesis (Table 11), where 0.1M xanthane hydride in pyridine was used as the sulfurization reagent and 20% DEA in ACN was used as an auxiliary wash post synthesis. All monomers (Table 12a) were made at 0.1M in ACN and contained a molecular sieves trap bag. The structure of linked cholesterol is shown in Table 12b.
The antisense strands were typically cleaved and deprotected (C/D) at 45° C. for 16-24 hours. The sense strands were typically cleaved and deprotected from the CPG using cold 50% (methylamine/ammonia hydroxide 28-30%) at ambient temperature for 2-3 hrs, whereas 3% DEA in ammonia hydroxide (28-30%, cold) was typically used for the antisense strands. C/D was determined complete by IP-RP LC/MS when the resulting mass data confirmed the identity of sequence. RNA hydroxy desilylation may be carried out using triethylamine trihydrofluoride in DMSO. Dependent on scale, the CPG was filtered via 0.45 um PVDF syringeless filter, 0.22 μm PVDF Steriflip® vacuum filtration or 0.22 μm PVDF Stericup® Quick release. The CPG was typically back washed/rinsed with either 30% EtOH/RNAse free water then filtered through the same filtering device and combined with the first filtrate. This was repeated twice. The material was then divided evenly into conical centrifuge tubes to remove organics via Genevac™. After concentration, the crude oligonucleotides were diluted back to synthesized scale with RNAse free water and filtered either by 0.45 μm PVDF syringeless filter, 0.22 μm PVDF Steriflip® vacuum filtration or 0.22 μm PVDF Stericup® Quick release.
The crude oligonucleotides were purified via AKTA™ Pure purification system using anion-exchange (AEX) or reverse-phase (RP) chromatography. For AEX, an ES Industry Source™ 15Q column with MPA: 20 mM NaH2PO4, 15% ACN, pH 7.4 and MPB: 20 mM NaH2PO4, 1M NaBr, 15% ACN, pH 7.4. For RP, an ES Industry Source™ 15RPC with MPA: 50 mM sodium acetate, 10% ACN and MPB: 80% ACN. Fractions which contained a mass purity greater than 85% without impurities >5% where combined.
The purified oligonucleotides were desalted using 15 mL 3K MWCO centrifugal spin tubes at 3500×g for ˜30 min. The oligonucleotides were rinsed with RNAse free water until the eluent conductivity reached <100 usemi/cm. After desalting was complete, 2-3 mL of RNAse free water was added then aspirated 10×, the retainment was transferred to a 50 mL falcon tube, this was repeated until complete transfer of oligo by measuring concentration of compound on filter via nanodrop. The final oligonucleotide was then nano filtered 2× via 15 mL 100K WCO centrifugal spin tubes at 3500×g for 2 m. Cholesterol-linked oligonucleotides were annealed at this stage to give cholesterol conjugated dsRNA by mixing equimolar aliquots of sense and antisense strands at room temperature for 30 minutes. The final desalted oligonucleotides were analyzed for concentration (nano drop at A260), characterized by IP-RP LC/MS for mass purity (Table 10) and UPLC for UV-purity.
Certain 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 controlled pore glass; “DAR” refers to drug/siRNA to antibody/protein ratio; “DCM” refers to dichloromethane; “DEA” refers to diethylamine; “DHAA” refers to dehydroascorbic acid; “DMSO” refers to dimethylsulfoxide; “DMT” refers to dimethoxytrityl; “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 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 spectrometer; “min” refers to minutes; “MW” refers to molecular weight; “MWCO” refers to molecular weight cut-off, “NHS” refers to N-hydroxysuccinimide; “OD” refers to optical density; “PBS” 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 reverse transcription-quantitative polymerase chain reaction; “SEC” refers to size exclusion chromatography; “siRNA” refers to small interfering RNA; “SMCC” refers to succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate; “SS” refers to sense strand; “TCO” refers to trans-cyclo-octene; “TfR” refers to transferrin receptor; “THF” refers to tetrahydrofuran; “TRIS” refers to tris(hydroxymethyl)aminomethane; “UPLC” refers to ultra performance liquid chromatography; and “UV” refers to ultraviolet.
SMCC-Functionalization of dsRNA
To a 50 mL conical tube containing amino-functionalized sense strand oligonucleotide SS-APP-AMINO, for example SEQ ID 172 appended to a C6-amino chain via a 3′-terminal phosphorothiolate ester, as a solution in water (8.42 mL, 0.023 mmol, 19.857 mg/mL), was added sodium bicarbonate powder (59 mg, 0.702 mmol). The mixture was briefly vortexed and sonicated to dissolve the bicarbonate. A freshly prepared solution of (2,5-dioxopyrrolidin-1-yl) 4-[(2,5-dioxopyrrol-1-yl)methyl]cyclohexanecarboxylate (96 mg, 0.281 mmol) in acetonitrile (6.32 mL) was then added to the bicarbonate-oligo solution, for example, dsRNA-48-PS-C6-amino (8.42 mL, 0.023 mmol, 19.857 mg/mL in water) and vortexed for 30 seconds. Then, the reaction was allowed to proceed for 4 hours with shaking at ambient temperature at 300 rpm, at which point temperature control on a ThermoMixer® C took the reaction mixture down to 10° C. for 15 hours. At this point, LTQ-MS analysis indicated full conversion. The reaction was quenched to pH 5 using 1N HC1 (621 μL, 0.621 mmol). The quenched reaction mixture was then concentrated to approximately ½ volume using a GeneVac™ centrifugal evaporator and the resultant precipitate-containing suspension was filtered using a 0.22 micron Steri-Flip® apparatus to remove precipitate, rinsing once with 5 mL of nuclease-free water. The resulting clear solution containing oligo was then diluted to approximately 55 mL with 20% acetonitrile in nuclease-free water and concentrated using a CentriCon® ultrafiltration apparatus (3000 MWCO regenerated cellulose membrane). Following passage of all the volume through the Centricon®, two more 55 mL portions of 20% acetonitrile in nuclease-free water were passed through the CentriCon® to rinse the material, and finally one passage of 55 mL pure Milli-Q® water to remove residual acetonitrile. The retentate was then recovered by inverting the Centricon® apparatus on the included recovery cup. The Centricon® apparatus was then washed and aspirated twice with 800 μL nuclease-free water in each of the two filtration pores (1.6 mL total per wash), and the combined rinsate and retentate were passed through a 50k MWCO filter, which was rinsed one more time with 5 mL nuclease-free water. Finally, the desired compound was measured for concentration using a NanoDrop™ apparatus (OD260—calculated extinction coefficient: 216.09 mmol-1 cm-1) to give the desired compound (SEQ ID 172 with appended C6-amino-SMCC) as a solution of 9.77 mg/mL in 13.219 mL (129 mg, 68.1%). LTQ-MS: observed deconvoluted m/z=7361.7, calculated mass 7361.17, mass purity 91.37%.
To a conical tube containing SS-APP-AMINO-SMCC, for example SEQ ID NO 172 with appended C6-Amino-SMCC (12.05 mL, 0.016 mmol, 1.328 mmol/L), was added its corresponding SS-APP-ANTISENSE, for example SEQ ID NO 173 with 5′-E-vinyl phosphonate, (0.0165 mmol, 2.619 mmol/L). The solutions were shaken at 25° C. for 30 minutes to give the desired SMCC-functionalized dsRNA (SMCC-dsRNA), then refrigerated to 10° C. for storage. The annealed solutions were sampled for LTQ purity and UPLC non-denaturing chromatography. Analysis via non-denaturing UPLC (run at 10° C.) shows a major single peak of 92% purity. LTQ-MS: (Antisense strand observed deconvoluted m/z=7768.4, calculated 7769.04; Sense strand observed deconvoluted m/z=7360.4, calculated mass 7361.17).
Conjugation Scheme for SMCC Functionalized dsRNA
The typical conjugation method utilized the SMCC-functionalized dsRNA for conjugating onto the engineered cysteine of the TfR binding proteins. For this method, TfR binding protein was prepared similarly as above to make the engineered thiol available for conjugation by undergoing a reduction and oxidation process of the TfR binding proteins. This was followed by incubating the SMCC-dsRNA with the TfR binding proteins at 4 molar equivalents for overnight conjugation at 4° C.
Optionally, following conjugation a maleimide hydrolysis step can be done to secure the linker-payload in terminal stage and avoid deconjugation during human body circulation via retro-Michael addition. This succinimide ring hydrolysis process was done by elevating the conjugate pH to 9.0 using 50 mM Arginine (stock solution of 0.7M arginine, pH 9.0 was used) and incubating the solution at 37° C. for 20 hours. The hydrolysis state of the maleimide was confirmed by LCMS characterization of +18 Da that is incurred by the water addition to the succinimide ring.
Synthesis of Mal-Tet-TCO and GDM linkers and conjugation of Mal-Tet-TCO- or GDM linker-functionalized dsRNA to the engineered cysteine of the TfR binding proteins have been described in WO 2024/036096.
Conjugation was monitored using analytical anion exchange chromatography. A ProPac™ SAX-10 HPLC Column, 10 μm particle, 4 mm diameter, 250 mm length was utilized with the following method. Flow rate of 1 mL/min, Buffer A: 20 mM TRIS pH 7.0, Buffer B: 20 mM TRIS pH 7.0+1.5M NaCl, at 30° C.
Drug/siRNA to antibody/protein ratio (DAR) was calculated based on peak area % from the analytical anion exchange (aAEX) chromatogram.
Post conjugation of dsRNA to the TfR binding protein, excess dsRNA and unconjugated protein was removed by further purification. Either preparative size exclusion chromatography (SEC) or preparative anion exchange chromatography was utilized for purification of the final conjugate. Preparative SEC was performed using Cytiva Superdex® 200 in 1×PBS pH 7.2 under an isocratic condition. Alternatively, anion exchange, e.g., ThermoFisher POROS™ XQ, was used with starting buffer of 20 mM TRIS pH 7.0 and eluting with 20 column volume gradient with a buffer containing 20 mM TRIS pH 7.0 and 1M NaCl. These resulted in purified TfR binding protein-dsRNA conjugate devoid of excess dsRNA and minimal unconjugated protein. The resulting conjugate profile was analyzed by analytical anion exchange for final DAR quantitation (Table 13).
In Vitro Potency Assessment of Cholesterol Conjugated dsRNTA Targeting APP in SHSY5Y and Mouse Cortical Neurons
Selected APP RNAi agents (cholesterol conjugated dsRNA targeting APP) were tested in vitro for APP inhibition in cultured SH-SY5Y cells and mouse cortical neurons.
SH-SY5Y cells (ATCC CRL-2266) were derived from the SK-N-SH neuroblastoma cell line (Ross, R. A., et al., 1983. J Natl Cancer Inst 71, 741-747). The base medium was composed of a 1:1 mixture of ATCC-formulated Eagle's Minimum Essential Medium, (Cat No. 30-2003), and F12 Medium. The complete growth medium was supplemented with additives including 10% fetal bovine serum. Cells were incubated at 37° C. in a humidified atmosphere of 5% CO2. On day one, SH-SY5Y cells were plated in fibronectin coated tissue culture plates and allowed to attach overnight. On day two, complete media was removed and replaced with RNAi agent in serum free media. Cells were incubated with RNAi agent for 72 hours before analysis of gene (mRNA) expression. RT-qPCR was performed to quantify targeted mRNA levels using TaqMan™ Fast Advanced Cell-to-CT kit following the manufacturer's protocol (ThermoFisher A35377). The delta-delta CT method of normalizing to a housekeeping gene, GAPDH (ThermoFisher, Hs99999905_m1, GAPDH; Hs00169098_m1, APP), was used to determine relative amounts of gene (mRNA) expression. A three or four parameter logistic fit was used to determine IC50.
Mouse primary cortical neurons were isolated from wild type C57BL6 mouse embryos at E18. On day 7, half of the medium was removed from each well and 2× concentration of RNAi agent in 2% FBS containing culture media with was added and incubated with cells for 7 days of treatment. At the end of treatment, RT-qPCR was performed to quantify targeted mRNA levels using TaqMan™ Fast Advanced Cell-to-CT kit. The delta-delta CT method of normalizing to a housekeeping gene, 3-actin probes (ThermoFisher, Mm02619580_g1, ACTB; Mm01344172_m1, APP), was used to determine relative amounts of gene (mRNA) expression. A three or four parameter logistic fit was used to determine IC50.
As shown in Tables 14A, 14B and 15, cholesterol conjugated dsRNA targeting the APP coding region (Tables 14A and 14B) or 3′UTR (Table 15) successfully reduce human APP gene (mRNA) expression in SHSY5Y cells and mouse cortical neurons. Table 16 shows the efficacy of cholesterol conjugated dsRNA targeting APP with different 2′-fluoro modification patterns of either the sense strand or the antisense strand.
Selected APP RNAi agents (TfR binding protein-dsRNA conjugates targeting APP) were tested in vitro for APP inhibition in EFO-21 cells and mouse cortical neurons (MCN).
EFO-21 cells (Simon, W. E., et al., 1983. J Natl Cancer Inst 70, 839-845) were derived from human ovarian carcinomas. The base medium was composed of RPMI supplemented with additives including 20% fetal bovine serum. Cells were incubated at 37° C. in a humidified atmosphere of 5% CO2. On day one, EFO-21 cells were plated in tissue culture plates and allowed to attach overnight. On day two, media was removed and replaced with RNAi agent and 1.5% serum containing media. Cells were incubated with RNAi agent for 72 hours before analysis of gene (mRNA) expression. RT-qPCR was performed to quantify targeted mRNA levels using TaqMan™ Fast Advanced Cell-to-CT kit following the manufacturer's protocol (ThermoFisher A35377). The delta-delta CT method of normalizing to a housekeeping gene, GAPDH (ThermoFisher, Hs99999905_m1, GAPDH; Hs00169098_m1, APP), was used to determine relative amounts of gene (mRNA) expression. A three or four parameter logistic fit was used to determine IC50.
Results provided in
Mouse primary cortical neurons were isolated from wild type C57BL6 mouse embryos at E18 and cultured as described above. On day 7, half of the medium was removed from each well and 2× concentration of dsRNA was added as either a cholesterol or antibody conjugated dsRNA (isotype antibody APP siRNA or mTBP1 antibody APP siRNA) in 2% FBS containing culture media was added and incubated with cells for 7 days of treatment. At the end of treatment, RT-qPCR was performed to quantify targeted mRNA levels using TaqMan™ Fast Advanced Cell-to-CT kit. The delta-delta CT method of normalizing to a housekeeping gene, 3-actin probes (ThermoFisher, Mm02619580_g1, ACTB; Mm01344172_m1, APP), was used to determine relative amounts of gene (mRNA) expression. A three or four parameter logistic fit was used to determine IC50.
Results provided in
In Vivo Potency Assessment of Cholesterol Conjugated dsRNA Targeting APP in Mouse after Single Intracerebroventricular (ICV) Dose
Selected APP RNAi agents (cholesterol conjugated dsRNA targeting APP) were also studied in wildtype C57BL/6N mice. Mice received ICV injection of 30 μg of the APP RNAi agent with different 2′-fluoro modification patterns (dsRNA No. 48 and dsRNA No. 63 in Table 7a) or PBS (phosphate buffered saline) and were sacrificed on Day 14 after the injection. Mouse APP mRNA expression in brain were measured and analyzed by qPCR, APP probe (Mm00431829_m1). The delta-delta CT method of normalizing used include housekeeping genes, 3-actin and GAPDH probes (Mm02619580_g1 and Mm99999915_g1, respectively). Protein expression was quantified using an immunoassay to assess Aβ(1-x), Aβ(1-40) and Aβ(1-42), peptide levels in homogenized brain tissues. Briefly, protein for Aβ peptide analysis was extracted from brain tissue using a Guanidine-HCL extraction protocol to capture both soluble and insoluble Aβ species. The assay used to detect Aβ(1-x) protein in brain homogenate was a standard sandwich enzyme-linked immunosorbent assay (ELISA) using commercially available or in-house generated antibodies and protein standards. Briefly, the capture antibody used was M266 (Haraln/Envigo) which recognizes Aβ(1-42) peptide aa 13-28 epitope. The detector 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). ELISA assays were developed with UltraTMB-ELISA substrate (Thermo Scientific). Analyzed data was normalized to total protein concentration of brain sample and reported as pg/mg brain.
The results shown in
In Vivo Potency Assessment of TfR Binding Protein-dsRNA Conjugates Targeting APP in Mouse and Cynomolgus Monkey after Single Peripheral IV Dose.
To demonstrate that TfR binding protein-dsRNA conjugates cross the blood brain barrier (BBB) to deliver dsRNA cargo to the CNS, studies were conducted on select APP RNAi agents to assess pharmacodynamic efficacy and corresponding brain exposure after peripheral delivery via an intravenous route of delivery. Specifically, human TfR transgenic knock-in mice where the extracellular domain of transferrin-receptor have been humanized, received a single 10 mg/kg (dsRNA) IV dose of human TfR binding proteins-dsRNA targeting APP conjugates TBP4-dsRNA No. 48 (DAR2) or TBP5-sdRNA No. 48 (DAR1), or a PBS (phosphate buffered saline) control. Animals were sacrificed 28 days after injection. Brain samples were collected to assess pharmacodynamic efficacy and tissue exposure. To measure brain tissue exposure timepoints between 0.25 and 672 h (28 days) post-dose were collected and conjugate-associated dsRNA levels (ng/g) were quantified by reverse phase LC/MS after antibody-enrichment via immunoprecipitation (IP RP LC/MS).
Results are shown in
To further demonstrate efficacy of TfR-shuttled conjugates, in vivo studies of dsRNA conjugates targeting different of APP CDS and 3′UTR sequences were also evaluated in the hTfR mouse. Table 17 reports reduction of APP mRNA levels (mean, n=4) in disease-relevant cortical and hippocampal regions 28 days after a single 1 mg/kg-IV bolus injection was achievable with multiple dsRNA targeting sequences.
The efficacy of selected TfR binding protein-dsRNA conjugates were further tested in Cynomolgus monkey (Macaca fascicularis). To assess the efficacy Cynomolgus monkeys (four per group) received a single injection of 10 mg/kg (effective dsRNA concentration) in the Saphenous vein of the thigh. The monkeys were injected with either PBS (phosphate buffered saline) or the APP RNAi agent TBP4-dsRNA No. 48 (DAR2) or TBP5-dsRNA No. 48 (DAR1) and sacrificed 29 days after dosing. Deeply anesthetized animals underwent cardiac perfusion, then brain, spinal cord and peripheral tissues were collected. The perfused brain was coronally sectioned, and punches were collected from subregions including prefrontal cortex, temporal cortex, motor cortex, parietal cortex, hippocampus and frozen. Additional tissues collected from spinal cord, liver, kidney, and muscles were also collected. To assess target mRNA and protein levels by RT-qPCR and -ELISA respectively in tissue homogenates. mRNA expression levels of human APP were quantified via a delta-delta CT method with GAPDH being used as the housekeeping gene for CNS regions. Further, conjugate-associated dsRNA level in the brain tissue (28-day terminal) and plasma (various time-points between 0-672 h) were assessed by IP RP LC-MS. Protein for Aβ peptide analysis was extracted from brain tissue using a Guanidine-HCL extraction protocol to capture both soluble and insoluble Aβ species. The assay used to detect Aβ(1-x) protein in brain homogenate was a standard sandwich enzyme-linked immunosorbent assay (ELISA) using commercially available or in-house generated antibodies and protein standards. Briefly, the capture antibody used was M266 (Haraln/Envigo) which recognizes Aβ(1-42) peptide aa 13-28 epitope. The detector antibody for cyno was an in-house generated biotinylated 3D6 human/Cyno Aβ(1-42) peptide aa 1-5 epitope antibody. The recombinant protein standard used for Cyno was human Aβ(1-40). ELISA assays were developed with UltraTMB-ELISA substrate (Thermo Scientific). Analyzed data was normalized to total protein concentration of brain sample and reported as pg/mg brain.
Assessment of the conjugate-associated dsRNA levels are presented in
An additional longitudinal study assessing the durability of the mRNA and protein reductions after a single 10 mg/kg IV dose in Cynomolgus monkey (3 animals per group) for human TfR binding proteins-dsRNA conjugate, TBP5-dsRNA No. 48., is presented in
Having demonstrated the potency of the human TfR binding proteins-dsRNA conjugates by intravenous route of administration, the efficacy of TBP5-dsRNA No. 48 (DAR1) conjugate delivered by a single subcutaneous (SC) administration and comparison to IV administration were studied.
Selected APP RNAi agents with different antisense strand modifications (e.g., dsRNA Nos. 107, 108, 109, 110 from Table 7a) were tested in vitro for inhibiting APP expression in EFO-21 cells. EFO-21 cell culture methods are described in Example 4.
Results provided in Table 18 show cholesterol conjugated dsRNA Nos. 107, 108, 109, and 110 successfully reduced APP gene (mRNA) expression in EFO-21 cells and their IC50.
Selected TfR binding protein-dsRNA conjugates with different dsRNA modifications were tested in vivo to assess pharmacodynamic efficacy after peripheral delivery via an intravenous route of delivery in both rodents and non-human primates. More specifically, human TfR transgenic knock-in mice where the extracellular domain of transferrin-receptor has 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 assess pharmacodynamic efficacy as shown in
The durability of the mRNA and protein reductions after a single 10 mg/kg IV dose was also performed in a Cynomolgus monkey (3-4 animals per group) for human TfR binding proteins-dsRNA conjugate, TBP5-dsRNA No. 109, is presented in
| Number | Date | Country | |
|---|---|---|---|
| 63578018 | Aug 2023 | US | |
| 63645219 | May 2024 | US |
