Lise Munsie, Ph.D., is Senior Development Manager and Project Leader, Pluripotent Stem Cell Therapies at CCRM, a Canadian, not-for-profit group that is focused on developing and commercializing regenerative medicine, and cell and gene therapy technologies.
1) Advances in Proteostasis Research
Decreased expression of proteasome activator in HD associated with increased aggregation
A hallmark of Huntington’s disease (HD) pathophysiology is the accumulation of aggregates of mutant huntingtin protein (mtHTT) in the affected neurons in the brains. mtHTT is initially soluble and aggregation is induced by the expanded poly-glutamine tract.
It is hypothesized that modulating levels of soluble mtHTT prior to aggregation could have therapeutic benefit. To achieve this therapeutically, research suggests increasing protein degradation through controlling the cells’ innate protein degradation systems (Proteostasis). There is data to support that modulating proteostasis has therapeutic benefits in models of HD through decreasing cellular aggregation of mtHTT.
A recent study looked at how components of the proteasome, a group of proteins in the cell responsible for protein degradation in Proteostasis, may regulate levels of soluble mtHTT and thus aggregation.1 There are a group of proteins called proteasome activators (PA) that allow proteins in and out of the proteasome. PA28 is one of these proteins, and is composed of two homologous subunits — PA28alpha and PA28beta (PA28αβ).
In this study, the group modulated the activity of PA28αβ in HD mouse models and found a decrease of functional PA28αβ in the parts of the brain affected by HD. The group found that while overexpression of PA28αβ can lead to an increase in the degradation of poly-glutamine peptides, mtHTT is too big for overexpression of PA28αβ to have an effect on it. However, silencing PA28αβ does lead to an increase in mtHTT aggregation. The group’s conclusion is that PA28αβ has an indirect effect on the mtHTT aggregation pathway.
NOVEL PROTEINS MODULATE MTHTT AGGREGATE FORMATION
Scientists can take cells from adult HD patients and reprogram them into stem cells, which are called induced pluripotent stem cells (iPSC). Stem cells are cells that have the ability to become any cell type in the body. By reprogramming HD patient cells into iPSC, scientists can then turn the iPSC into neurons and study actual neurons from the patient.
Interestingly and importantly, there is no aggregation of mtHTT in neurons derived from HD patient stem cells (HD-iPSC).
A group hypothesized that the reprogramming process may cause changes to the proteostasis systems, which is why these cells do not have aggregates. Therefore, they set out to assess what is different about HD-iPSC neurons on a molecular level to see if this can be leveraged in drug discovery.2 Using iPSC derived from control and HD patients, the group used a technique called co-immunoprecipitation to identify proteins that HTT interacts within these neurons.
The group identified a novel protein that interacted with HTT in this model — Ras GTPase-activating protein-binding protein 1 (G3BP1). G3BP proteins are part of a cellular structure called stress granules, which are aggregates that form during stress and are meant to protect the proteins in the aggregates from the stressor. Once the stress is removed, these aggregates are meant to dissociate. During aging or in disease, these granules can inappropriately persist.
The group found that iPSC express high levels of GBP31 that decrease during differentiation to neurons, and knocking down GBP31 led to the induction of aggregation of mtHTT in HD-iPSC. They confirmed these results in a worm model of poly-glutamine expansion, finding that knock-down of GBP31 led to increased aggregation.
They found that mtHTT interacts with soluble G3BP1 and that during stress G3BP1 forms stress granules. When in this form, they cannot interact with mtHTT. In these circumstances, mtHTT is also more likely to form aggregates, which supports that soluble G3BP1 can inhibit aggregate formation and that stress to cells is likely to lead to increased mtHTT aggregation due to lack of available G3BP1.
Finally, using another cellular model, the group over-expressed G3BP1 and found that the over-expression led to a decrease in the ability of mtHTT to aggregate, and the group showed this was through G3BP1 activity in the Proteostasis pathway.
This work defines how stress granule formation, G3BP1 and related Proteostasiss pathways are involved in HD, which is important for understanding disease and drug discovery pathways.
1 Geijtenbeek, K.W. et.al. (2022) Reduction in PA28αβ activation in HD mouse brain correlates to increased mHTT aggregation in cell models. Plos One: https://doi.org/10.1371/journal.pone.0278130
2 Gutierrez-Garcia, R. et.al. (2023) G3BP1-dependent mechanism suppressing protein aggregation in Huntington’s models and its demise upon stress granule assembly. Human Molecular Genetics: https://doi.org/10.1093/hmg/ddac304
2) CRISPR Research for HD Editing
CRISPR-Cas9 silences mtHTT gene, attenuates aggregation
The most accepted therapeutic approach to slow or stop the progression of Huntington’s disease (HD) is elimination of mutant huntingtin gene expression (mtHtt). The current strategy being utilized in clinical trials is administering antisense oligonucleotide (ASO) to lower gene expression, leading to a subsequent decrease in protein expression. The ASO can either be non-specific and silence both wildtype (wtHtt) and mtHtt or, preferably, allele-specific, silencing only mtHtt.
The main caveat with ASO is that it only transiently silences gene expression, which means that it must be continuously administered to a patient throughout their lifetime. Disappointingly, current clinical trial data is not supporting efficacy or safety of the ASO approach, which means different strategies to accomplish silencing should be examined.
A powerful new technology that allows for elegant and specific genetic manipulation is the CRISPR-Cas9 system. This system can be delivered to cells, produces targeted double-stranded breaks in DNA, and endogenous repair mechanisms that often lead to permanent gene silencing at the targeted area. This system is being investigated in treating different diseases where alterations to gene expression could lead to positive outcomes.
A recent study tested the ability for CRISPR-Cas9 to target the CAG repeat in the Htt gene.1 The group used the HD R6/2 mouse model and produced neurospheres from the brains of these mice. The CRISPR-Cas9 system targeting either the DNA sequencing upstream or downstream of the CAG repeat areas in the Htt gene, was delivered to the neuropheres.
After delivery, the group assessed Htt aggregation — the hallmark of mtHtt expression in this model — and found that delivery of CRISPR-Cas9 attenuated aggregation. The group also assayed for levels of BDNF and PGC-1a, two proteins known to be decreased in pathological HD, and found increased expression levels of both in the treated neurospheres. The overall outcome was higher cell viability and a decrease in cell death, indicating that the CRISPR-Cas9 system can be used to edit mHtt, leading to positive outcomes.
Although there are potential benefits to using CRISPR-Cas9 to edit DNA, the major caveat is that the change cannot be modulated or reversed if there are ill-intended consequences. Thus targeting RNA in an allele-specific manner is still desirable.
There is an RNA-targeting subtype of CRISPR-Cas, VI-D CRISPR-Cas Cas13d, which has RNase activity and can cleave target RNA. This system was recently exploited for testing in HD models.2 The group made a Cas13d selective for expanded CAG (Cas13d-CAGEX). They used adeno-associated virus-mediated delivery of Cas13d-CAGEX in several models of HD, including zQ175/+ HD mice, neurons derived from HD patient-induced pluripotent stem cells, and fibroblast lines derived from HD patients.
Cas13d-CAGEX resulted in allele-selective suppression of mtHtt, with limited off-target effects and long-lasting efficacy for a variety of clinically relevant CAG repeat lengths.
In the mouse models, there were additional phenotypic readouts such as decreased aggregation and increased brain volume when Cas13d-CAGEX was delivered compared to a control vector, indicating that the knock-down levels were efficacious for changing the course of pathogenesis. Cas13d-CAGEX is a new method for allele-specific silencing that may prove to be safer and more efficacious than ASO.
1 Han, J.Y et al. (2022) CRISPR-Cas9 mediated genome editing of Huntington’s disease neurospheres. Molecular Biology Reports: https://doi.org/10.1007/s11033-022-08175-6
2 Morelli, K.H et al. (2022) An RNA-targeting CRISPR-Cas13d system alleviates disease-related phenotypes in Huntington’s disease models. Nature Neuroscience: https:// doi.org/10.1038/s41593-022-01207-1
3) Seeking HD Biomarkers
Proteins in cerebral spinal fluid potential biomarkers of HD progression
One major challenge impeding the success of clinical trials is a lack of reliable biomarkers to track disease onset or progression. A biological molecule that may be present in bodily fluids, like blood, that tracks with HD progression and is not be found at the same levels in a healthy individual, would be ideal for clinical trials. Multiple studies seek to develop biomarkers for HD in easy-to-obtain bodily fluids like cerebral spinal fluid (CSF), blood, or skin.
CSF is thought to be an ideal biomarker for HD, as it specifically reflects changes occurring in the brain. A recent hypothesis-driven study used a very sensitive technique called nanoflow liquid chromatography-coupled parallel-reaction monitoring mass spectrometry (nanoLC-PRM-MS) to test 26 proteins in the CSF of control, pre-HD and man-HD patients.1 Some of the 26 proteins have previously been implicated as affected in HD and others were exploratory.
The results from this study confirmed changes in some previously reported markers in HD CSF as well as identifying some new CSF proteins that track with disease. It is unlikely that a single marker will track with HD, and as such, a panel of markers may be required. This study suggests different panels of markers can discern the different groups of patients.
Proteins PENK, ALB and NEFL can discriminate between control patients and pre-HD, which makes this panel good for timing interventions prior to onset. Proteins PP1R1B, TTR, CHI3L1 and CTSD can discern pre-HD from early onset, and proteins CNR1, PP1R1B, BDNF, APOE and IGHG1 can discern early stage from late-stage HD, giving researchers a toolbox of proteins to track during intervention.
The technique used in this study, nanoLC-PRM-MS, is a very sensitive and specific technique, however, it requires expensive equipment not practical for clinical sampling. Therefore, future work should focus around developing assays to track these proteins from CSF using more conventional immunoassays.
MicroRNA AS BLOOD-BASED BIOMARKERS
Although CSF is an excellent source of biomarkers for neurodegenerative disease due to the correlation with the brain, peripheral blood is an easier and less invasive fluid to sample, and therefore finding markers of disease that track in blood would be of great benefit to clinical trials. One candidate for biomarkers is microRNA — non-coding cellular RNA fragments that circulate in the blood and have been investigated in the past as biomarker candidates.
Recently, a group investigated a different class of non-coding small guide RNA, called small nucleolar RNA (snoRNA), as a potential biomarker candidate for HD that can be detected in peripheral blood.2 The group performed a microarray study from whole noncoding RNA expression profiles from the plasma of blood of HD patients and controls, which included healthy subjects and psychiatric patients. The study showed a significant increase in the plasma levels of a specific snoRNA, SNORD13 in HD patients, and an additional difference in levels of SNORD13 between pre-HD and controls.
Confirmatory studies were done, which further included Alzheimer’s disease patients, showing the changes in circulating SNORD13 were specific to HD and tracked with the natural history of HD progression. As an easily measurable, inexpensive and quantifiable test, further longitudinal validation of SNORD13 in HD patient cohorts could help further qualify this snoRNA as a biomarker for HD.
1 Caron, N.S et al. (2022) Cerebrospinal fluid biomarkers for assessing Huntington disease onset and severity. Brain Communications: https://doi.org/10.1093/braincomms/fcac309
2 Romano, S. et al. (2022)Circulating U13 Small Nucleolar RNA as a Potential Biomarker in Huntington’s Disease: A Pilot Study. International Journal of Molecular Sciences: 23:12440