by Jena Berndt
Angelman syndrome Acronym Legend
AAV-GT: Adeno-associated Virus- gene replacement therapy
ASO: antisense oligonucleotides
ATF-ZF: Artificial Transcription Factors/Zinc Fingers
CAS9: CRISPR associated protein 9
CAS13: CRISPR associated protein 13
CRISPR: clustered regularly interspaced short palindromic repeats
ERT: Enzyme Replacement Therapy
gRNA: guide RNA
HSC-GT: Hematopoietic Stem Cell- gene replacement therapy
RNAi: RNA interference
SNA: spherical nucleic acids
shRNA: short-hairpin RNA
UBE3A: Human UBE3A gene or RNA
UBE3A: Human UBE3A protein (ubiquitin ligase)
UBE3A-ATS or UBE3A-AS: Human UBE3A antisense transcript (RNA)
ube3a: rodent ube3a gene or RNA
ube3a-ATS or ube3a-AS: rodent ube3a antisense transcript (RNA)
ube3a: rodent ube3a protein
Let’s break down the various therapeutic approaches currently being advanced for the potential treatment of humans living with Angelman syndrome. These include Adeno-associated virus gene replacement therapy (AAV-GT), Hematopoietic Stem Cell gene replacement therapy (HSC-GT), antisense oligonucleotides (ASO), spherical nucleic acids (SNAs), Artificial Transcription Factors/Zinc Fingers (ATF-ZF), Enzyme Replacement Therapy (ERT), clustered regularly interspaced short palindromic repeats (CRISPR), short-hairpin RNA (shRNA), micro-RNA (miRNA), and various downstream targets that improve the cellular message, generally at the synapse, or the communication between neurons.
I will break each approach down to help us all better understand what each one means for our loved ones living with Angelman syndrome.
One approach, AAV-GT, involves delivering a healthy copy of the missing or non-functional UBE3A gene directly into brain cells, called neurons. This allows these neurons to make the protein that is either missing or not functioning properly, the ubiquitin ligase protein (UBE3A). There are different ways to deliver this type of therapy. This can be through an injection into the cerebral spinal fluid (CSF) (e.g., down by the lower back (lumbar puncture) or up at the base of the skull (ICM; intracisterna magna)), directly into the tissue of the brain (intraparenchymal), or through a vein (intravenous). The veins cannot deliver most AAVs for brain disorders as these virus’ do not generally cross the blood vessels (blood-brain barrier) to get into the neurons very well, which is where we need them to be. There are currently at least 7 AAV-GT programs in the pipeline, and 4 are being robustly funded by FAST.
A second approach, HSC-GT, involves putting the UBE3A gene into a patient’s own blood stem cells, which are young cells we all have in our bone marrow that are able to grow, or differentiate, into any type of blood cell because they are so young. These cells can be removed through a blood draw (leukapheresis), and the modified UBE3A gene can be added to those cells outside of the patient. Then, those cells can be injected back into the patient through their vein, and they can incorporate back into the bone marrow so that all of the different blood cell lines (e.g., red blood cells, white blood cells, platelets, etc.) contain the new UBE3A gene. Once these cells are within the bone marrow they then travel around the bloodstream, and can cross the blood-brain barrier. Once inside the brain they sit next to the neurons (microglial cells) in the brain and release the this new UBE3A gene that the neurons of those with AS are missing. The modifications to this inserted gene are made so that neurons can take it up throughout the brain. This is called cross-correction, where UBE3A can be secreted and taken up by many neurons in the area. There is currently one HSC-GT program in the pipeline, and this is fully FAST funded.
A third approach, ASOs or SNAs, involves using a combination of synthetic RNA and DNA to bind to the RNA of the UBE3A-ATS (antisense transcript), which is responsible for silencing the paternal copy of the UBE3A gene in neurons. These drugs are designed to be very specific in where they bind to the UBE3A-ATS and aim to unsilence the paternal UBE3A gene. There are currently at least 4 programs taking this approach in the pipeline, 1 of which is FAST funded.
A fourth approach, ATF/ZF, involves aiming to bind the UBE3A-ATS in a specific promoter area, resulting in unsilencing of the paternal UBE3A gene. FAST has been supporting this research at UC Davis.
A fifth approach, CRISPR, consists of two components the CAS enzyme that can cut DNA (CAS9 or CRISPR associated protein 9) or RNA (CAS13) and a guide RNA (gRNA) that can recognize specific sequences of DNA or RNA to cut. For Angelman syndrome, CRISPR-CAS would involve finding the sequence of the human genome that could permanently inhibit the UBE3A-ATS from silencing the paternal UBE3A gene. This is delivered into the brain using an AAV, as mentioned above for AAV-GT but instead of replacing the human UBE3A gene it would carry the guide (gRNA) to inhibit the UBE3A-ATS. Ensuring we can get this to enough neurons to have robust paternal UBE3A expression is key. The gRNA is then attached to the DNA cutting enzyme (CAS9), or RNA cutting enzyme (CAS13), and introduced to the neurons. After this, they can either modify, delete, or insert new sequences to that sequence specific stretch of the DNA or RNA. You can think of this as a cut and paste tool. There are currently at least 3 programs in the pipeline, 2 of which are FAST funded.
A sixth approach, ERT, could provide the missing UBE3A enzyme as a purified protein directly to the brain, and this would be modified to enter neurons and function inside and outside the cells. There is currently 1 program in the pipeline, which is FAST funded.
A seventh approach, shRNA/miRNA, stands for short hairpin RNA and microRNA, and this is also termed “RNA Interference” or RNAi. Both of these vector (AAV) introduced approaches to target the UBE3A-ATS and activate the silent paternal copy of the UBE3A gene in neurons. There are currently 3 programs in the pipeline, 1 of which is FAST funded.
An eighth approach includes an entire category of downstream targets, focusing on different molecular pathways and effector proteins impacted by the missing UBE3A protein. These drugs generally aim to improve the communication of neurons at the synapse (junction between 2 neurons). There are currently at least 5 different drug candidates in the pipeline, 4 of which are FAST funded. Overall, when we think about a pipeline of at least 8 different approaches, including 25 programs, being evaluated to potentially treat Angelman syndrome, we are hugely optimistic that the future for all individuals living with AS can be far different than what we were told on diagnosis day. We are proud that FAST has taken the initiative to robustly support 16 of these 25 programs in order to de-risk them toward human clinical trials. We will continue to advance the most promising technology and science forward for human benefit.