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CRISPR is everywhere, CRISPR is fascinating! Let's take a quick refresher before we dive into the paper, shall we?

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Intro to Cas9 gene editing technology (DC)

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Intro to Cas9 gene editing technology (DC)

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A short refresher on CRISPR-Cas9 for those who may need it!

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"Okay, but what about actual symptoms?" (DC)

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"Okay, but what about actual symptoms?" (DC)

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Figure 3: Can SaCas9 gene therapy vector rescue phenotypes in AS mice?

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

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Body weight in female mice, clasping time, centre time for open field test showed complete rescue to WT levels (Figures 3b,d,e)!

They saw significant improvements on brain weight, rotarod test, but not quite wildtype levels. Rotarod test results were maintained until week 28 (Figures 3c,h,i).

There were no improvements on distance travelled in open field test, marble burying test (Figures 3f,g).

AS mice didn't show any deficits in contextual and cue based learning and memory tasks, and they were unable to evaluate rescue of cognitive deficits.

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

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Figure 4: How is Ube3a-ATS silenced via cas9 vector?

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.

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“With great gRNAs comes great CRISPR therapies” (AR)

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“With great gRNAs comes great CRISPR therapies” (AR)

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The authors designed 260 Staphylococcus pyogenes Cas9 (SpCas9) gRNAs that target putative regulatory regions and genes around Ube3a-ATS.

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.

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Q1: What gRNA can be used to unsilence paternal Ube3a?

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?

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Forward direction:

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.

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To summarize how the gene therapy vector disrupts ATS.. (DC)

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To summarize how the gene therapy vector disrupts ATS.. (DC)

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In the forward orientation, gene trap of Ube3a-ATS causes premature transcription termination at AAV derived polyA sequence.

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!

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Good science means being critical, so let's discuss some concerns and strengths!(AR)

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Good science means being critical, so let's discuss some concerns and strengths!(AR)

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Good news first, so the strengths :

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.

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Cas9 gene therapy for Angelman Syndrome in humans could be available to us in the near future, thanks to papers like this laying the groundwork and providing insights enabling the development of human gene therapy!

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A roughly $40 million National Institutes of Health (NIH) grant awarded to Yale School of Medicine will support the development of a gene-editing platform technology capable of reaching the human brain. The innovative new genome-editing technology, which was developed from the first phase of the NIH Common Fund Somatic Cell Genome Editing (SCGE) program, could potentially lead to treatments or cures for many neurogenetic diseases.

If we can prove the concept of this technology in the two diseases we're studying, we can then apply it to hundreds or thousands of diseases of the brain.

Yong-Hui Jiang, MD, PhD

Neurogenetic disorders can be devastating, and treatments are scarce. The two-phase grant from the NIH will support research into a novel CRISPR-based gene-editing technology and delivery platform for targeting neurogenetic diseases. The grant will focus on Angelman syndrome and H1-4 syndrome as a proof-of-concept, and could be applicable to many neurogenetic disorders. The new delivery technology, known as STEP (Stimuli-responsive Traceless Engineering Platform), has the capacity to revolutionize genome-editing therapy and create a one-time treatment for a range of genetic disorders.

The work will be led by Yong-Hui Jiang, MD, PhD, professor of genetics, of pediatrics and of neuroscience and chief of medical genetics, and Jiangbing Zhou, PhD, professor of neurosurgery and of biomedical engineering, both at Yale School of Medicine. The team also includes co-leader Elizabeth Berry-Kravis, MD, PhD, professor of pediatrics and neurological sciences and director of the RUSH Pediatric Neurosciences F.A.S.T. Center for Translational Research at RUSH University in Chicago; and Allyson Berent, DVM, DACVIM and Jennifer Panagoulias of the Foundation for Angelman Syndrome Therapeutics (FAST). The HIST1H1E foundation led by Kimberly Greenberg will also participate the study. The first $26.5 million grant will support pre-clinical and toxicology studies in animal models and human-brain organoids. If the milestones of the first phase are met, the NIH will provide an additional $13 million to fund clinical trials in humans.

"If we can prove the concept of this technology in the two diseases we're studying, we can then apply it to hundreds or thousands of diseases of the brain," says Jiang.

Existing gene therapy methods face limitations

Until now, the development of gene-therapy technologies has been slow as researchers focus on one disease at a time. Furthermore, existing gene therapies commonly rely on viruses as the delivery vehicle, but this method has risks. The exposure of the body to viral proteins can trigger adverse reactions. Viruses can incorporate DNA into the human genome and increase risk for tumor development. Viruses may be particularly not ideal for delivery of genome-editing therapy due to a risk of off-target effects associated with long-term gene expression. While non-viral nanoparticle-based delivery platforms for genome editing options do exist, their delivery to the brain has been challenging due to many limitations.

New platform utilizes ribonucleoproteins (RNPs) that directly access the brain

At Yale, a joint team led by Jiang and Zhou invented the STEP technology and successfully applied it for delivery of genome editing therapy for treatment of various diseases in animal models. Instead of viruses, the novel platform utilizes a chemical engineering approach to achieve brain-wide delivery of gene-editing with high efficiency.

"Over the past few years, we have been working on development of this technology. I am thrilled about the opportunity to bring it to the clinical bedside," says Zhou.

Through this new system, the team administers the delivery technology intrathecally, or through a spinal tap. Due to their unique physical chemical properties, STEP-RNPs demonstrate a great ability to penetrate the brain and edit neuronal cells with high efficiency. After entering the cells, RNPs are quickly degraded, limiting the risk of off-target effects.

Other molecular therapies such as antisense oligonucleotides (ASOs) require frequent treatments, but this new technology could work as a single dose with a permanent effect and potentially with a greater efficiency than ASOs.

A potential therapeutic platform for a range of brain diseases

The team will be focused on Angelman syndrome and H1-4 syndrome because prior research already indicates significant promise of the benefits of gene-editing therapy in treating these conditions. But ideally, their strategy can be adopted for treating many other neurogenetic disorders as well. In particular, some neurogenetic conditions are rare diseases, so there is often little incentive for pharmaceutical companies to invest in therapeutics. The researchers hope their platform will increase these companies' interest in investing because it can be applied to many different diseases.

In May, Yale School of Medicine joined the NORD Rare Disease Centers of Excellence network. This work adds to a growing concentration of groundbreaking basic research, clinical translation, and clinical care in the rare disease space taking place at Yale School of Medicine.

Other faculty participants for the projects from Yale School of Medicine include James McPartland, PhD; Julie Wolf, PhD; Michele Spencer-Manzon, MD; Hui Zhang, MD, PhD; Caroline Hendry, PhD; James Dziura, PhD, MPH; Nigel Bamford, MD; Kathleen Cardinale, MD; Caihong Qiu, PhD; and Eric Velazquez, MD. The NIH Federal Award Identification Number for the grant is UG3TR004713.

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Large NIH Grant Supports CRISPR-based Gene Therapy Development for Brain Diseases

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The two-phase grant will support research into a novel CRISPR-based gene-editing technology and delivery platform for targeting neurogenetic diseases.

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Self-generated figure to explain the experimental steps for testing gRNAs for unsilencing paternal UBE3A[Biorender]

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“So many gRNAs, now time to test them”(AR)

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“So many gRNAs, now time to test them”(AR)

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They did a co-staining experiment and for this, they take cortical neurons from a mice that has a knock-in YFP reporter at the paternal UBE3A locus. Then they transfect these neurons with 3 things : cam2ka promoter driving td Tomato expression in neurons, the gRNA and then the cas9.

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"Mirror, mirror on the wall, which gRNA is the best of them all?" (AR)

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"Mirror, mirror on the wall, which gRNA is the best of them all?" (AR)

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In this figure, black circles are measuring the percentage of neurons expressing YFP (signal UBE3A expression). The fold change here is greater than 4 between treated and WT neurons.

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!

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Here, we are looking at results from RT-qPCR quantification after they transduced Ube3am+/pYFP cortical neurons with lentivirus for SpCas9 + Spjw33

Main takeaways

-Spjw33 gRNA effectively un-silences paternal UBE3A expression

-Specific against the Snord115 and promoter regions.

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Cool! We see UBE3A expression in mice cortical neurons, but what about throughout the brain?(AR) 

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Cool! We see UBE3A expression in mice cortical neurons, but what about throughout the brain?(AR) 

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Here, they took a similar gRNA to Spjw33 (Sajw33) and Cas9 under a neuronal promoter and packaged them into AAV9. They injected this AAV9 vector intracerebroventricularly into mice at the postnatal day 1 (P1) stage. These mice were the typical AS mouse model with (Ube3am−/p+) - maternally mutated and paternally silenced  They then immunostained for UBE3A and NEUN at P90.

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.

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Nice visuals, but show me the graphs!(AR)

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Nice visuals, but show me the graphs!(AR)

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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.

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Angelman Syndrome is a genetic disorder that results in sympotoms such as developmental delays, intellectual disabilities, extensive speech impairment, issues with movement and balance, epilepsy, and abnormal sleep-wake.

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Here's a couple of future experiments they can conduct:

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).

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Angelman Syndrome results from the loss of function of the maternal copy of the Ube3a gene located on chromosome 15. The Ube3a gene encodes an E3 ubiquitin ligase, an enzyme with many roles. It marks proteins for degradation, behaves as a transcriptional coactivator, and is involved with synaptic function and neuroplasticity.

The paternal copy of Ube3a is silenced by Ube3a-ATS via genomic imprinting and only the maternal copy is meant to be viable. The loss of function of the maternal copy leading to Angelman can be from large deletions, truncating or missense mutations in the gene, paternal isodisomy (two paternal copies of the UBE3A gene), or imprinting defect (maternal copy is epigenetically altered to look like the paternal copy).

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Rather than trying to salvage the loss of the maternal copy, unsilence the paternal copy!

Ube3a-ATS, a noncoding RNA silences Ube3a via methylation. So if we silence the silencer.. then we can get Ube3a back!

Use AAV to deliver CRISPR-Cas9 to Ube3a-ATS

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1. Wolter JM, Mao H, Fragola G, Simon JM, Krantz JL, Bazick HO, Baris Oztemiz, Stein JL, Zylka MJ. 2020. Cas9 gene therapy for Angelman syndrome traps Ube3a-ATS long non-coding RNA. Nature. 587(7833):281–284. doi:https://doi.org/10.1038/s41586-020-2835-2.

2. CheckRare. 2020. Angelman Syndrome Explained. YouTube. [accessed 2023 Dec 9]. https://www.youtube.com/watch?v=2dPEmXhJHcc&ab_channel=CheckRare.

3. Imprinting and Genetic Disease: Angelman, Prader-Willi and Beckwith-Weidemann Syndromes | Learn Science at Scitable. 2014. Naturecom. [accessed 2023 Dec 11]. https://www.nature.com/scitable/topicpage/imprinting-and-genetic-disease-angelman-prader-willi-923/.

4. Yamaguchi H, Honda S, Torii S, Shimizu K, K. Katoh, Miyake K, Miyake N, Nobuhiro Fujikake, Hajime Tajima Sakurai, Arakawa S, et al. 2020. Wipi3 is essential for alternative autophagy and its loss causes neurodegeneration. Nature Communications. 11(1). doi:https://doi.org/10.1038/s41467-020-18892-w.

5. Xhako E. 2020 Oct 23. Snapshot: What is the Rotarod Test? National Ataxia Foundation. [accessed 2023 Nov 21]. https://www.ataxia.org/scasourceposts/snapshot-what-is-the-rotarod-test/.

6. Dorninger F, Gundacker A, Zeitler G, Berger J. 2019 Aug 13. Ether Lipid Deficiency in Mice Produces a Complex Behavioral Phenotype Mimicking Aspects of Human... ResearchGate. [accessed 2023 Nov 27]. https://www.researchgate.net/publication/335147704_Ether_Lipid_Deficiency_in_Mice_Produces_a_Complex_Behavioral_Phenotype_Mimicking_Aspects_of_Human_Psychiatric_Disorders.

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Abidur Rahman: AR

Dominique Cho: DC