RNA editing

RNA Editing

Adenosine Deaminase Acting on RNA (ADAR)

  • ADARs were discovered due to their ability to deaminate adenosines in long, double-stranded RNA, converting them to inosines.
  • ADARs are expressed in human cells.
  • The ADAR family includes ADAR1, ADAR2 (ADARB1), and ADAR3, but only ADAR1 and ADAR2 have demonstrated catalytic activity.
  • A-to-I deamination is the most common type of RNA editing in mammals, and defects in this process are linked to human diseases like central nervous system disorders and pediatric astrocytomas.
  • Circular ADAR-recruiting guide RNAs (cadRNAs) enable efficient programmable adenosine-to-inosine RNA editing without exogenous proteins.
  • cadRNAs facilitate robust and durable RNA editing across multiple sites and cell lines and high transcriptome-wide specificity.
  • Incorporating interspersed loops in the antisense domains increases transcript-level specificity for the target adenosine, reducing bystander editing.
  • In vivo delivery of cadRNAs via adeno-associated viruses enabled 53% RNA editing of the mPCSK9 transcript in C57BL/6J mice livers and 12% UAG-to-UGG RNA correction of the amber nonsense mutation in the IDUA-W392X mouse model of mucopolysaccharidosis type I-Hurler syndrome.
  • cadRNAs enable efficient programmable RNA editing in vivo with diverse protein modulation and gene therapeutic applications.
  • In vivo repair of a guanosine-to-adenosine mutation in methyl CpG binding protein 2 RNA that causes the neurodevelopmental disease Rett syndrome. Repair is mediated by hippocampal injections of juvenile Mecp2^{317G>A} mice with an adeno-associated virus expressing the hyperactive catalytic domain of adenosine deaminase acting on RNA 2 and Mecp2 guide. After 1 month, 50% of Mecp2 RNA is recoded in three different hippocampal neuronal populations. MeCP2 protein localization to heterochromatin is restored in neurons to 50% of wild-type levels. Whole-transcriptome RNA analysis of one neuronal population indicates that the majority of off-target editing sites exhibit rates of 30% or less.
  • Rett syndrome is caused by de novo loss-of-function mutations in the gene encoding X-linked transcriptional regulator MECP2
  • MeCP2 pathological mutations result primarily in a neurological phenotype characterized in females by regression of speech and purposeful hand motions, seizures and respiratory abnormalities
  • MeCP2 is expressed in most, if not all, neurons and glia
  • Mouse line in which the mouse Mecp2 gene contained the human patient mutation MECP2^{317G>A} (R106Q)
  • AAV PHP.B-ADAR2 technology to edit the mutation
  • Muscular dystrophies (MD) are a genetically and clinically heterogeneous group of rare neuromuscular that cause progressive weakness and breakdown of skeletal muscle over time.
  • The disorders differ as to which muscles are primarily affected, the degree of weakness, how fast they the disease burden is.
  • Some types are also associated with problems in other organs.
  • Over 30 different disorders are classified as muscular dystrophies – one of those, Duchene Muscular Dystrophy (DMD) accounts for approximately 50% of cases and affects males beginning around the age of four.
  • This team developed an AAV8 mediated ADAR RNA editing therapy for a DMD mouse model, mdx.
  • Mdx mouse model - which bears an ochre stop site in exon 23 of the dystrophin gene.
  • This choice was additionally motivated by the fact that nonsense mutations are responsible for nearly 11% of all described gene lesions causing inheritable human disease, and close to 20% of disease- associated single base substitutions that affect the coding regions of genes
  • AAV8, and injected 2E+122E+12 vector genomes (vg)/muscle into the tibialis anterior (TA) or gastrocnemius of mdx mice.
  • Targeted the mdx mice via CRISPR-Cas9 based excision of exon 23
  • Ornithine transcarbamylase deficiency (OTCD) is an X-linked genetic disorder
  • Prevents the breakdown and excretion of ammonia; this allows ammonia to rise to toxic levels and affect the central nervous system.
  • The organ most involved in the processing of ammonia is the liver.
  • The urea cycle converts ammonia into urea, which is water soluble and readily excreted in the urine
  • Current drug and surgical treatments are often ineffective due to the severity of the disease
  • Team developed an AAV8 mediated ADAR therapy using a mouse model of OTC with a point mutation.
  • Spfash mice harbour a G>A point mutation in the last nucleotide of the fourth exon of the OTC gene, which leads to OTC mRNA deficiency and production of a mutant protein

Cas13

  • CRISPR systems uncovered a novel type of RNA targeting enzyme, Cas13.
  • Cas13s function similarly to Cas9, using a ~64 -nt guide RNA to encode target specificity.
  • The Cas13 protein complexes with the guide RNA via recognition of a short hairpin in the crRNA, and target specificity that is complementary to the target region.
  • In addition to programmable RNase activity, all Cas13s exhibit collateral activity after recognition and cleavage of a target transcript, leading to non -specific degradation of any nearby transcripts regardless of complementarity to the spacer.
  • Cas13a showed some activity for RNA knockdown, certain orthologs of Cas13b proved more stable and robust in mammalian cells for RNA knockdown and editing.
  • More recently, additional orthologs of Cas13 have been discovered, including Cas13d, which has been leveraged for efficient and robust knockdown across many endogenous transcripts
  • Studies showed that Cas13d can be used to modulate splicing of endogenous transcripts and that the coding sequence for Cas13d is small enough to fit within the packaging limits of AAV for in vivo delivery.
  • Frontotemporal Dementia with Parkinsonism linked to Chromosome 17 (FTDP -17) is an autosomal dominant major neurodegenerative disease caused by diverse point mutations in MAPT, the gene encoding for tau.
  • Tau exists as two major isoforms in human neurons, 4R and 3R
  • Some FTD are caused by mutations in the intron following MAPT exon 10 and elevate the expression of 4R tau
  • dCas13 used to target to MAPT exon 10 to alleviate dysregulated 4R/3R tau ratios.
  • Patient -derived human induced pluripotent stem cells (hiPSCs) were differentiated into cortical neurons
  • AAV1 carrying dCas13 that target the exon 10 splice acceptor
  • dCasRx-mediated exon exclusion was able to reduce the relative 4R/3R tau ratio by nearly 50% relative to a vehicle control
  • Suggesting that dCas13 can be exploited for transcriptional modulation in primary cell types via AAV delivery.
  • Huntington’s disease (HD) is a fatal, dominantly inherited neurodegenerative disorder caused by CAG trinucleotide expansion in exon 1 of the huntingtin (HTT) gene.
  • Developed a mutant allele -sensitive CAGEX RNA -targeting CRISPR –Cas13d system (Cas13d – CAGEX) that eliminates toxic CAGEX RNA in fibroblasts derived from patient with HD and induced pluripotent stem cell - derived neurons
  • Tested in vivo in a heterozygous HD mouse model using AAV9 via intra -striatum
  • RNA Editing for Programmable A to I Replacement (REPAIR), works by fusing the ADAR2 deaminase domain to Cas13b
  • Two types of REPAIR 1 or 2
  • REPAIRv1 had many off-targets due to its high activity and overexpression
  • Studies showed by generating mutants in the ADAR2 catalytic site, we could lower the off - targets by two orders of magnitude and still retain on -target editing (10%10\%-40%40\% editing) with REPAIRv2.
  • Although v2 had a lower efficiency than v1
  • An alternative approach for lowering the off - target rate is to decrease the expression of the Cas13 -ADAR2 fusion while keeping guide expression high.
  • Most Cas13 proteins studied to date are around 130 kDa in size, and even the smallest member of the family (Cas13d) is 100 kDa
  • Large size is challenging for packaging into a translational viral vector for direct protein delivery
  • People have circulating antibodies to the CRISPR-Cas proteins
  • Targeting RNA therapeutically with continuously delivered microbially derived effector proteins could eventually lead to substantial immunogenicity challenges.
  • Thus, led to development of CRISPR-Cas inspired RNA targeting system (CIRTS)

CIRTS

  • CIRTS is not only smaller than naturally occurring CRISPRCas programmable RNA binding systems
  • BUT can also be built entirely from human protein parts.
  • CIRTS provides a potential strategy to avoid immune issues when applied to epitranscriptome- modulating therapies.

Conclusion

  • Overview of the different types of RNA editing
  • Technology is continually changing
  • Proof of concept studies in vitro and in vivo showing the editing abilities