Patricia F. Fitzpatrick Dimond Ph.D. Technical Editor of Clinical OMICs President of BioInsight Communications

Lack of Effective In Vivo Animal and In Vitro Models Stymie More Effective Research

Scientists continue to progress toward the goal of using human stem cells to treat incurable human diseases. But these cells, particularly neural stem cells, may also provide models for a variety of disorders, including psychiatric, neurodegenerative, and cardiovascular.

To date, a dearth of representative models for many of these diseases has impeded the discovery and development of effective therapeutics. The most common adult neuroeurodegenerative disorders, Alzheimer’s Disease (AD) and Parkinson’s Disease (PD), have proven particularly difficult to model either in animals or in vitro. Most drugs entering the AD drug development pipeline have failed; only one agent specifically for AD has received approved over the past 10 years—Axona (caprylidene) from Accera, approved in March 2009—for an overall candidate drug failure rate of about 99.6%.

Recently, induced pluripotent cell lines (iPSC) lines have been generated from skin cells of individuals with a variety of degenerative diseases, including AD and PD. Investigators say that when a disease-specific phenotype is detectable in differentiated cells, reprogramming technology provides a new opportunity to identify aberrant disease-associated pathways and drugs that can block them. By studying the differences between normal and diseased cells, researchers can better understand what causes cells to die and identify key points in the process that may lead to treatments to change the course of the disease.

Some of the first diseases modeled with iPSCs have been neurodegenerative that affect children (e.g., Retts Syndrome, or RTT, a severe X-linked neurodevelopmental disorder that affects 1 in 10,000–20,000 girls worldwide, making it one of the most common forms of mental retardation in females). A key discovery in 1999 identified a causative link between mutations in the methyl-CpG binding protein, MeCP2, and RTT, thereby enabling mechanistic studies and providing a target for potential treatments.

MeCP2 protein functions as a transcriptional repressor critical for normal neurological function. Prior studies demonstrated that either loss or doubling of MeCP2 results in postnatal neurodevelopmental disorders; restoration of MeCP2 function in a mouse model of the disease reverses the neurological symptoms in adult mice raising the possibility that this disorder may be treatable in humans.

Maria C.N. Marchetto, Ph.D., and colleagues working at The Salk Institute, UCSD, and Penn State, developed a human model of RTT by generating iPSCs from fibroblasts of RTT patients carrying different MeCP2 mutations and unaffected individuals. The investigators reported that the RTT-iPSCs retained the capacity to generate proliferating neural progenitor cells (NPCs) and functional neurons that underwent the X-inactivation typical of differentiated cells in females.

In the iPSC neurons, they observed a reduced number of dendritic spines and synapses in iPSC-derived neurons and detected an altered frequency of intracellular calcium spikes and electrophysiological defects in RTT-derived neuronal networks. Their data indicate a potential imbalance in the neuronal networks associated with RTT pathology, according to the scientists, and their findings provide valuable information for RTT and, potentially, autistic spectrum disorder (ASD) patients, since they suggest that presymptomatic defects may represent novel biomarkers to be exploited as diagnostic tools and that early intervention may be beneficial.

Last April, Bristol-Myers Squibb (BMS) announced that it had acquired iPierian, a biotech company focused on the discovery and development of new treatments for tauopathies, which represent a class of neurodegenerative diseases associated with the pathological aggregation of Tau protein in the human brain.

IPierian has developed its monoclonal antibody program based on Tau discoveries made using the company’s iPSC technology. The company used iPSC disease models that combine human cortical neurons, motor neurons, microglia, and astrocytes in a dish to discover and validate novel therapeutic targets or mechanisms of disease. Leveraging the company's iPSC capabilities, the company said, can provide insight into the earliest drivers of diseases such as Alzheimer's, in contrast to conventional autopsy samples that typically only allow for study of end-stage pathophysiology.

The acquisition gives BMS full rights to iPierian’s lead preclinical monoclonal antibody, IPN007, that may offer a promising new approach to treat progressive supranuclear palsy and other tauopathies, and that could potentially enter Phase I clinical trials this year.

The antibody, BMS says, represents a new approach to Alzheimer’s by targeting a novel form of secreted Tau protein that is differentially regulated in Alzheimer’s disease patients to slow the spread of Tau throughout the brain and, therefore, inhibit the associated disease progression

Parkinson’s Disease

In most individuals, PD occurs idiopathically, arising sporadically with no known cause. However, about 15% of individuals have family members with Parkinson's disease. By studying families with hereditary Parkinson's disease, scientists have identified several genes that are associated with the disorder, resulting in the discovery of three genes and the mapping of five others that are linked to rare familial forms of the disease (FPD).

The products of the identified genes, including α-synuclein (PARK 1), parkin (PARK 2), and ubiquitin-C-hydrolase-L1 (PARK 5), remain the subject of intense studies designed to elucidate the underlying mechanism of FPD pathogenesis. Particularly noteworthy was the discovery reported in 2006 in The New England Journal of Medicine of an “astonishing high” prevalence of a single mutation in leucine-rich repeat kinase 2 (LRRK2), G2019S, in North African Arabs and Ashkenazi Jews with PD.

While the function of the LRRK2 protein is not yet fully determined, it occurs in brain areas most affected by PD. The G2019S mutation is believed to be responsible for upregulation of LRRK2 kinase activity, which may ultimately play a role in neuronal loss. The authors noted that the G2019S mutation appeared to be an important cause of both familial and sporadic Parkinson's disease in this group of Ashkenazi Jewish individuals.

Several laboratories have developed iSPC models from individual PD patients. These models, the scientists say, have the potential to enable characterization of the complex pathophysiologic mechanisms underlying the disease, allowing identification of potential interventions earlier in its course.

Because PD is characterized by loss of A9 dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc), investigators have generated iPSC-derived midbrain dopaminergic neurons to investigate pathogenic mechanisms in cell type as a means of modeling PD.

Byers et al. working at Stanford University reported the generation of iPSC-derived midbrain dopaminergic neurons from a patient with a triplication in the α-synuclein gene (SNCA). They observed that the iPSCs readily differentiated into functional neurons, and the PD-affected line exhibited disease-related phenotypes in culture. These included α-synuclein accumulation, inherent overexpression of markers of oxidative stress, and sensitivity to peroxide-induced oxidative stress.

They concluded, based on these findings, that the dominantly acting PD mutation can intrinsically perturb normal cell function in culture and confirmed that these features reflect, at least in part, a cell autonomous disease process that is independent of exposure to the entire complexity of the diseased brain.

Other investigators, in a further refinement of iPSC neurodegenerative modeling, say that the immaturity of neurons differentiated from human induced pluripotent stem cells presents difficulties for modeling late-onset neurodegenerative disorders such as Parkinson’s disease.

The investigators say this raises concerns as to how well iPSC-derived cells can model diseases where patients do not develop symptoms until later in life, implicating age as a necessary component to disease progression. They note that several studies of these cells have shown loss of age-associated characteristics during iPSC induction, including increase in telomere length, loss of mitochondrial fitness, and loss of senescence markers in iPSCs derived from older donors.

Justine D. Miller and colleagues working at Memorial Sloan Kettering, Boston Children’s Hospital, Weill Cornell Medical College, DNAVEC, and other institutions developed a strategy for inducing age-related features based on Progerin expression in iPSC-derived fibroblasts and neurons. Progerin, a mutated version of the normal cellular protein lamin A, functions to maintain the structure of a cell’s nucleus. A truncated form of the protein is a hallmark of the early aging disease, Progeria.

The investigators found that by use of synthetic mRNA to overexpress progerin they could reestablish age-related markers in iPSC-fibroblasts, including dopamine-specific phenotypes such as neuromelanin accumulation. Induced aging in PD iPSC-derived dopamine neurons revealed disease phenotypes that require both aging and genetic susceptibility, such as pronounced dendrite degeneration, progressive loss of tyrosine hydroxylase (TH) expression, and enlarged mitochondria or Lewy-body-precursor inclusions. Thus, they concluded, their study suggests that progerin-induced aging can be used to reveal late-onset age-related disease features in hiPSC-based disease models.

As investigators continue to refine iPSC models for degenerative diseases, the possibility of developing drugs to prevent these disorders, halt their advancement, and even reverse them may be closer to realization. 

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