Journal: Gut

Article Title: RNA-based therapies in liver metabolic diseases

doi: 10.1136/gutjnl-2023-331742

Figure Lengend Snippet: Current RNA therapeutics for metabolic monogenic disorders. ASOs and siRNAs are complementary oligonucleotides that target specific mRNAs through Watson-Crick base pairing, while exogenous mRNAs are delivered to cells to produce the desired proteins. CRISPR-Cas9, a gene-editing tool, disrupts disease-causing genes at specific locations via an sgRNA guide with a sequence that is complementary to the target gene. CRISPR leverages the Cas9 protein as ‘molecular scissors’ to cut DNA, activating a cellular repair process that can either disrupt the faulty gene, silence it, or permanently correct deleterious mutations to restore proper protein expression. There are multiple Cas proteins with distinct functions. While Cas9 remains the most widely used for genome editing, Cas12, Cas13, and Cas14 expand CRISPR’s applications to RNA editing, disease detection, and more precise DNA modifications, respectively. ASO, antisense oligonucleotide; CRISPR-Cas9, clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9; mRNA, messenger RNA; sgRNA, single guide RNA; siRNA, short interfering RNA.

Article Snippet: Fabry disease , X-linked , Childhood/ Teenagehood , Glycolipids (GLA) , Lysosome , Gal A mRNA , Moderna Translate Bio , Zhu et al 2019 DeRosa et al 2019 , .

Techniques: CRISPR, Sequencing, Expressing, Small Interfering RNA

Journal: Gut

Article Title: RNA-based therapies in liver metabolic diseases

doi: 10.1136/gutjnl-2023-331742

Figure Lengend Snippet: Strategies for targeting siRNA and mRNA therapies to hepatocytes. (A) Delivery and mechanism of targeted silencing by siRNAs. Targeted delivery of siRNA to hepatocytes is achieved through chemical conjugation with a trimer of GalNAc, which avidly binds to the ASGPR that is predominantly expressed on liver hepatocytes. On cellular entry, the siRNA is processed by DICER, which discards the passenger strand and incorporates the guide strand into the RISC complex. The guide strand then directs the RISC to complementary mRNA sequences, where AGO2, an endonuclease within the RISC, cleaves the target mRNA, effectively silencing the target gene and preventing protein synthesis. (B) Delivery and mechanism of action of therapeutic mRNAs. LNPs are currently the most effective nanocarrier system for liver-targeted therapies following systemic administration. LNPs include amino ionisable cationic lipids, cholesterol, polyethylene glycol-lipid conjugates, helper phospholipids (phosphatidylcholines) and active mRNA molecules. Owing to the negative charge of mRNA, encapsulation is often achieved via electrostatic interactions with cationic lipids. An exchange of proteins from other lipid particles results in ApoE being present in LNPs, which facilitates hepatic LNPs uptake via LDLRs, highly abundant on the surface of hepatocytes. Additionally, delivery systems that incorporate GalNAc-lipid modified LNPs allow for LDLR-independent hepatocyte targeting via the ASGPR. On cellular uptake, LNPs become protonated in acidic environments, facilitating endolysosomal escape and mRNA release into the cytosol to be translated into the therapeutic protein. Proteins may remain within hepatocytes at their specific subcellular locations or may be secreted into the circulation to reach other target organs. ApoE, Apolipoprotein E; AGO2, argonaut 2; ASGPR, asialoglycoprotein receptor; GalNAc, N-acetylgalactosamine; LDLR, low-density lipoprotein receptor; LNPs, lipid nanoparticles; RISC, RNA-induced silencing complex; siRNAs: small interfering RNAs.

Article Snippet: Fabry disease , X-linked , Childhood/ Teenagehood , Glycolipids (GLA) , Lysosome , Gal A mRNA , Moderna Translate Bio , Zhu et al 2019 DeRosa et al 2019 , .

Techniques: Conjugation Assay, Encapsulation, Modification

Journal: Gut

Article Title: RNA-based therapies in liver metabolic diseases

doi: 10.1136/gutjnl-2023-331742

Figure Lengend Snippet: Structure of optimised mRNAs for therapeutic applications. (A) Structure of therapeutic mRNAs, including the five functional elements: 5′ cap, 5′ UTR, ORF, 3′ UTR and poly(A) tail. The 5′ cap protects mRNA from exonucleases and enables ribosome recognition. Optimised cap structures enhance translation and also reduce immune activation by recognition via RLRs. In the ORF, chemical modifications of nucleobases such as pseudouridines (Ψ), N1-methyl-pseudouridine (N1Ψ) or methylated nucleobases such as m1A, m6A, and m5C, and GC-rich sequences can be introduced to reduce immunogenicity, prevent unwanted secondary structures and increase protein expression. The 5’ UTR can be modified to increase translation by placing Kozak sequences from genes such as globin or Hsp70, and the modification of the 3’ UTR by including synthetic sequences from the albumin or Hsp70 genes can improve mRNA stability. UTRs can also be customised to enhance tissue-specific translation. The length of the poly(A) tail is also optimised to improve the synthetic capability of a given mRNA. (B) saRNAs use the self-replication basis of RNA alphaviruses to amplify RNA transcripts in the cytosol. They include two ORFs, one for the viral nsp proteins (1-4), which enable RNA replication and amplification within the cell increasing protein expression from small doses, and the ORF of the protein of interest. Another alternative to mRNAs is circRNA, which is devoid of a 5’ cap and a 3’ poly(A) tail, has a self-splicing intron (group I intron) to promote circularisation and an engineered IRES to drive protein synthesis. Compared with linear mRNAs, circRNAs exhibit reduced immunogenicity and enhanced stability leading to prolonged duration of translational activity compared to linear mRNAs. Ψ, pseudouridine; circRNA, circular mRNA; IRES, internal ribosome entry site; m5C, 5-methylcytosine; m6A, N6-methyladenosine; N1Ψ, N1-methyl-pseudouridine; NSP, non-structural protein; ORF, open reading frame; RLRs, retinoic acid-inducible gene I (RIG-I)-like receptors; saRNA, self-amplifying mRNA; 3' UTR, 3' untranslated region.

Article Snippet: Fabry disease , X-linked , Childhood/ Teenagehood , Glycolipids (GLA) , Lysosome , Gal A mRNA , Moderna Translate Bio , Zhu et al 2019 DeRosa et al 2019 , .

Techniques: Functional Assay, Activation Assay, Methylation, Immunopeptidomics, Expressing, Modification, Amplification, Activity Assay