Huntington’s disease

Axol Bioscience HD iPSC collection

Patient-derived iPSC lines allow the development of physiologically relevant cellular models with clear disease phenotypes for target identification and drug screening. iPSC lines derived from patient cells contain the CAG repeat disease mutations and the genetic background which contributes to disease onset and severity. Therefore, these lines can be used for models that enable the screening of potential genetic modifiers that are part of the “genetic background” that may impact disease onset, progression, and symptoms. Genetic modifiers themselves are also molecular targets for drug discovery.

Axol Bioscience has created five iPSC lines from materials donated from HD patients and one from an asymptomatic carrier. Each line undergoes rigorous quality control such as assessments of morphology, karyotype, pluripotency, sterility and CAG repeat sizing.

Cell line name	Disease	Disease status	Sex	Age	Gene	Confirmed genetic mutation
CENSOi019-B	Huntington’s Disease	Affected	F	7	HTT	CAG14/CAG127	Increasing CAG repeats
CENSOi019-A	Huntington’s Disease	Affected	F	7	HTT	CAG14/CAG127
CENSOi052-A	Huntington’s Disease	Affected	F	16	HTT	CAG28/CAG66
CENSOi017-A	Huntington’s Disease	Affected	F	51	HTT	CAG17/CAG42
CENSOi011-D	Huntington’s Disease	Affected	F	40-50	HTT	CAG18/CAG39
CENSOi053-A	Huntington’s Disease	Asymptomatic Carrier	M	64	HTT	CAG17/CAG38

Axol human iPSC lines derived from both asymptomatic and HD patients (Table 1) are differentiated into HD-relevant brain cell types, including striatal neurons, astrocytes, and microglia. These models enable the study of neuronal and neuroinflammatory processes play a role in HD pathogenesis.

 

Striatal neurons, astrocytes and microglial cells can be combined in vitro to develop complex physiologically relevant co-culture models to better understand and replicate disease phenotypes in vitro, and study brain regional differences in the disease process, as well as the cell-cell dependencies involved in HD-associated neurodegeneration.

The CENSOi019-B line demonstrates CAG repeat instability

The donor for the CENSOi019-B line was a 7-year-old female patient with severe, early onset HD. Genotypic analysis showed a CAG14/CAG125 genotype.

Further study suggested that this line has an atypical allele associated with hastened onset and progression of the disease.

The line proved to have instability in CAG repeat length with the number of repeats growing over time. After reprogramming and generation of the master bank, the number of CAG repeats was 144.

CAG expansion analysis during 15 passages showed an increase of 1 x CAG repeat every 5 passages. Whole Genome Sequencing (WGS) data from this iPSC line suggests the loss of the CAA interruption and presence of certain SNPs/INDELS in the mutant HTT allele that can be disease modifiers, driving somatic instability, age of onset and HD progression. Striatal neurons derived from this iPSC line exhibit CAG repeat instability, a hallmark of HD. This instability is critical for studying disease progression, as CAG repeat expansion contributes to neuronal dysfunction and degeneration. To better replicate HD pathology in vitro, iPSC-derived cells are used to assess morphology as well as functional endpoints such as trinucleotide instability, neurite outgrowth and spontaneous firing. Other endpoints can be explored, such as mitochondrial function and protein aggregation. As a result of these characteristics, the CENSOi019-B line and striatal neurons derived from this line have become a vital resource to the HD research community. The study of repeat expansion dynamics provides an in vitro model for early-onset HD, which is often more severe and less understood than the adult-onset form of the disease.

Importantly, to HD research, the CAG repeat number is unstable in both the iPSC and the derived striatal neurons, offering the opportunity to conduct ‘instability’ studies over time.

While the CENSOiO19 -B iPSCs line expands on average 1X CAG repeat every 5 passages (0.5X CAG/ week) the expansion in differentiated striatal neurons is slower rate with an average CAG repeat expansion of 0.13 × CAG/week.

To accelerate this process, researchers have investigated the modulation of FANCI-associated nuclease 1 (FAN1) expression level within these iPSC-derived striatal neurons. In the clinical setting, FAN1 is associated with delayed age at onset (AAO) and slower progression of HD, suggesting that FAN1 is protective in the context of an expanded HTT CAG repeat.

In preliminary studies, we have oberved ~80% knock-down of FAN1 at day 20 and conducted long range studies that resulted in accelerated expansion.