Subjects: Ube3am-/p+ (AS model) mice, WT mice

• Behavioural tests and physical measurements were performed over 40 weeks after SaCas9 gene therapy vector injection into AS mice to observe improvements in the Key symptoms of AS: microcephaly, deficits in proprioception and motor function and increased body weight with age.

• Behavioural Tests:

  • Hindlimb clasping - motor coordination

  • Accelerating rotarod - motor coordination and learning

  • Open field - locomotor activity and anxiety levels

  • Marble burying - anxiety and compulsive behaviour

  • Fear conditioning - learning and memory

• Physical Measurements: Brain weight, Body weight

In utero reactivation of patUbe3a can effectively treat many symptoms of AS! But not all..

• A series of primers were designed for detection of fusion RNAs between Ube3a-ATS and the AAV vector, which allows them to see where and how the vector inserts into the gene.

• Forward direction - Figure 4a

  • 3a. For/Rev primers targeting Ube3a-ATS 3 -> Ube3a-ATS (positive control)

  • 3b. Forward primer for SaCas9, Reverse primer for 3ŔACE anchor -> Ube3a-ATS:AAV:pA fusion transcripts!

• Reverse direction - Figure 4b

  • They used a Primer walking strategy - 1 forward primer for ATS + reverse primers that anneal progressively further into the AAV genome.

Q2: Does AAV delivery of SaCas9 and gRNA unsilence paternal Ube3a throughout the nervous system?

Q3: Can AAV delivery of SaCas9 and gRNA rescue phenotypes?

Q4: How does cas9 vector silence Ube3a-ATS?

• Fusion RNAs were only detected in Sajw33 treated animals. No fusion RNAs were detected in -taq (negative control) or Ube3a-ATS (positive control) mice.

• Based on the primer design they could conclude that fusion transcripts contain polyA from the 3’UTR of SaCas9, and this causes premature transcription termination of Ube3a-ATS!

Reverse direction:

• No fusion transcripts were detected beyond SaCas9.

• This suggests that convergent RNA Polymerase II transcription blocks Ube3a-ATS, which is a similar mechanism as paternal Ube3a/Ube3a-ATS collision model!

  
  

We now know that AAV integration can effectively disrupt Ube3a-ATS in distinct mechanisms.

In the reverse orientation, convergent transcription of Ube3a-ATS and cas9 transcript blocks Ube3a-ATS.

It's interesting to note that paternal Ube3a is silenced by convergent transcription of Ube3a/Ube3a-ATS in untreated AS as well!

a) They have conducted multiple orthogonal assays and model systems (e.g. cortical neurons, fluorescence studies, western blot, and multiple functional assays). It adds more confidence to their results!

A couple of weaknesses!

a) They could not show long-range and long-term rescue of Ube3a protein in the cerebellum.

b) AS model mice were unable to replicate cognitive deficits seen in AS patients, and the authors were entirely unable to investigate the rescue of cognitive deficits in the paper. Since intellectual disability is a major symptom of Angelman Syndrome, the inability to analyze this aspect is a major limitation of this paper.

Note, lpw is the long promoter region of the gene and Snord115 & Snord116 cluster are small nucleolar RNAs.

They skipped SNORD116 genes as disturbing this region causes Prader–Willi syndrome, so our best gRNA shud come from the other regions, including SNORD115.

The positive control here is topotecan that inhibits topoisomerase I, forms these stalled huge cleavage complexes and blocks  the lncRNA from inhibiting UBE3A. No more inhibition from lcRNA = UBE3A expression.  

The key takeaway is  the anti-SNORD115 gRNA, Spjw33 was very effective at unsilencing UBE3A allele.  They found their target!

Main takeaways

-Spjw33 gRNA effectively un-silences paternal UBE3A expression

-Specific against the Snord115 and promoter regions.

The figures speak for themselves : we see long-term and stable expression of paternal UBE3A as a result of our specific gRNA and CRISPR therapy. 

Western blot quantifications show us that paternal UBE3A protein was restored to 37% (cerebral), 38%(hippocampus) and 40% (spinal cord) of wild-type levels. Note that it was not significant for cerebellum.

So far, we can say that the CRISPR-Cas9 therapy does result in long-term and mostly, long-range expression of paternal UBE3A.

a) More quantitative and sensitive approaches? In order to probe why the cerebellum was not significantly rescued, they can conduct quantitative mass spectrometry. This approach bypasses antibody-related background and also can read absolute protein levels.

b) Some of their experiments involved a Cam2ka promoter, which is a generalized neuronal promoter. They can target specific brain regions and cell subsets with more specific promoters (e.g. glia in the cerebellum).

c) They can also take more multi-omic approaches where they can understand how their CRISPR-Cas9 therapy affects global transcriptomic and proteomic changes (e.g. using RNA-seq).

Dominique Cho: DC