Figure 3.
Targeting RNA in hepatocytes. (A) After IV administration, double strand siRNA encapsulated in a lipid nanoparticle, that is, patisiran and is opsonized by apoliprotein E (ApoE), goes to the liver through vascular fenestrations, and binds to ApoE receptors on hepatocytes surface. The antisense strand of the siRNA then enters an enzyme complex called the RNA–inducing silencing complex. The siRNA antisense strand binds to the same sequence within the 3′ untranslated region of both mutant and wtTTR mRNAs, and this enzyme complex subsequently binds to its complementary mRNA target sequence, mediating its cleavage by argonaute-2 endonuclease (Ago2), thereby preventing synthesis of the target protein ATTRv and ATTRwt (pathway 1). Another approach uses a siRNA conjugated to a trimer of Gal-NAC residues, that is, vutrisiran, which is recognized by and transported into hepatocytes by asialoglycoprotein receptors (ASGPRs) located on the surface of hepatocytes. After the ligand-receptor complex is internalized, the cargo is released into the endocytic pathway with subsequent engagement with the RNA-induced silencing complex (RISC). ASGPR is then rapidly recycled to the cell surface, thus enabling multiple rounds of cargo uptake and release (pathway 2). Second-generation ASO inotersen selectively targets liver TTR mRNA, hybridizing to the 30-nontranslated regions that contain no known TTR mutations. Inotersen binds to wt and mutant transthyretin RNA transcripts, resulting in their degradation in the nucleus by ribonuclease H. As a result, inotersen inhibits the production of wtTTR and all mutant forms of TTR (pathway 4). Eplontersen is conjugated to a triantennary GalNAc3 moiety to facilitate delivery to hepatocytes through ASGPR-mediated uptake (pathway 3). (B) TTR gene editing a TTR-specific single guide RNA (sgRNA) and a Cas9 mRNA encapsulated in a lipid nanoparticle is opsonized by ApoE, goes to the liver through vascular fenestrations, and binds to ApoE receptors at hepatocytes surface. Cas 9 mRNA is then translated in the cytoplasm, and the Cas9-sgRNA ribonucleoprotein enters the nucleus and provokes targeted DNA cleavage. As a result, endogenous DNA repair induces the appearance of indels in the TTR gene, leading to frameshift mutations impairing functional TTR protein production.

Targeting RNA in hepatocytes. (A) After IV administration, double strand siRNA encapsulated in a lipid nanoparticle, that is, patisiran and is opsonized by apoliprotein E (ApoE), goes to the liver through vascular fenestrations, and binds to ApoE receptors on hepatocytes surface. The antisense strand of the siRNA then enters an enzyme complex called the RNA–inducing silencing complex. The siRNA antisense strand binds to the same sequence within the 3′ untranslated region of both mutant and wtTTR mRNAs, and this enzyme complex subsequently binds to its complementary mRNA target sequence, mediating its cleavage by argonaute-2 endonuclease (Ago2), thereby preventing synthesis of the target protein ATTRv and ATTRwt (pathway 1). Another approach uses a siRNA conjugated to a trimer of Gal-NAC residues, that is, vutrisiran, which is recognized by and transported into hepatocytes by asialoglycoprotein receptors (ASGPRs) located on the surface of hepatocytes. After the ligand-receptor complex is internalized, the cargo is released into the endocytic pathway with subsequent engagement with the RNA-induced silencing complex (RISC). ASGPR is then rapidly recycled to the cell surface, thus enabling multiple rounds of cargo uptake and release (pathway 2). Second-generation ASO inotersen selectively targets liver TTR mRNA, hybridizing to the 30-nontranslated regions that contain no known TTR mutations. Inotersen binds to wt and mutant transthyretin RNA transcripts, resulting in their degradation in the nucleus by ribonuclease H. As a result, inotersen inhibits the production of wtTTR and all mutant forms of TTR (pathway 4). Eplontersen is conjugated to a triantennary GalNAc3 moiety to facilitate delivery to hepatocytes through ASGPR-mediated uptake (pathway 3). (B) TTR gene editing a TTR-specific single guide RNA (sgRNA) and a Cas9 mRNA encapsulated in a lipid nanoparticle is opsonized by ApoE, goes to the liver through vascular fenestrations, and binds to ApoE receptors at hepatocytes surface. Cas 9 mRNA is then translated in the cytoplasm, and the Cas9-sgRNA ribonucleoprotein enters the nucleus and provokes targeted DNA cleavage. As a result, endogenous DNA repair induces the appearance of indels in the TTR gene, leading to frameshift mutations impairing functional TTR protein production.

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