A Phase 0 biomarker study in patients with Parkinson*s Disease and healthy controls

Parkinson*s disease is the second most common neurodegenerative disease,
affecting approximately 1*2% of individuals aged 65 or over (de Rijk et al.
1997; Blin et al. 2015), and the prevalence is projected to increase
substantially as the population ages (Dorsey et al. 2007). Currently approved
treatments improve motor symptoms but do not address the underlying cause of
the disease. Over time, these symptomatic therapies lose effectiveness and are
associated with increasing frequency and severity of adverse effects, such as
dyskinesias and hallucinations. In addition, nonmotor symptoms, including
depression, anxiety, sleep disorders, cognitive impairment, and dementia, are
disabling and common features of Parkinson*s disease, but are poorly addressed
by current therapies (Aarsland et al. 1996; Truong et al. 2008; Lyons and Pahwa
2011; Khoo et al. 2013; US FDA *Voice of the Patient* 2016). As such,
Parkinson*s disease patients inevitably experience mounting disabilities over
the years to decades that they live with the disease (Hely et al. 2005). Thus,
there is a significant need for an effective disease-modifying therapy to
prevent the progressive motor and nonmotor disabilities not addressed by
current therapies.
LRRK2 mutations are an established cause of Parkinson*s disease, accounting for
approximately 4*5% of familial Parkinson*s disease (Healy et al. 2008; Chai et.
al. 2013). Familial LRRK2 mutations are transmitted in an autosomal dominant
pattern of inheritance with incomplete penetrance (Marder et al. 2015). In
addition, variants within the LRRK2 gene are a genetic risk factor and account
for 1*2% of sporadic Parkinson*s disease cases (Healy et. al. 2008; Chai et al.
2013; Hernandez et al. 2016). The majority of identified LRRK2 pathogenic
mutations are located within the central catalytic domain of the protein, and
these mutations increase kinase activity in in vitro assays (West et al. 2007;
Sheng et al. 2012).
LRRK2 encodes a multidomain protein containing a GTPase domain, a kinase
domain, and several potential protein-protein interaction domains. The majority
of identified pathogenic mutations in LRRK2 are located within its catalytic
domains, including the most common mutation associated with LRRK2-PD, G2019S.
These mutations increase LRRK2 kinase activity, either through direct mutations
within the kinase domain, or through indirect mechanisms. While the exact
pathogenic mechanisms remain unknown, LRRK2 is believed to play a role in
intracellular trafficking in the endo-lysosomal system (Henry et al. 2015;
Cookson 2016), and LRRK2 mutations with increased kinase activity result in
lysosomal dysfunction. Kinase activity is increased with LRRK2 mutations in
vitro, and cellular data shows that 50% inhibition of G2019S mutant LRRK2
activity in cells reverses the lysosomal abnormalities. Evidence of this effect
is further supported by data (on file with Denali Therapeutics Inc.) showing
that expression of fluorescently tagged G2019S LRRK2 in H4 neuroglioma cells
produces enlarged lysosomes, suggesting that increased LRRK2 kinase activity
disrupts the lysosomal pathway. This effect is dependent on LRRK2 kinase
activity, as treatment with LRRK2 kinase inhibitors related to DNL151 rescues
the observed defects in lysosomal morphology. Hence, inhibiting increased LRRK2
kinase activity may mitigate LRRK2 mediated pathogenesis, including lysosomal
dysfunction, supporting the therapeutic potential of DNL151 (Khan et al. 2005;
Jaleel et al. 2007; West et al. 2007; Sheng et al. 2012; Steger et al. 2016).
The role of LRRK2 in intracellular trafficking in the endo-lysosomal system
(Cookson 2016) is further supported by the fact that 1) expression of LRRK2
mutations with increased kinase activity results in altered lysosomal phenotype
and function, and 2) expression of LRRK2 mutations with increased kinase
activity (but not kinase-dead LRRK2 mutations) alters cell function and health
in cellular and in vivo models. Alterations in both lysosomal function and
cellular function associated with these LRRK2 mutations are reversed with LRRK2
kinase inhibitors (Henry et al. 2015; Lee et al. 2010).
LRRK2-driven Parkinson*s disease is characterized clinically by features
consistent with idiopathic Parkinson*s disease. The pathological features of
both forms of Parkinson*s disease are also similar, suggesting common
pathogenic mechanisms. Lysosomal dysfunction may be a central mechanism for
accumulation of intracellular proteins resulting in accumulation of *-synuclein
and the formation of Lewy bodies, a cardinal pathological feature of idiopathic
Parkinson*s disease (Dehay et al. 2013). The role of LRRK2 in *-synuclein
accumulation and consequent pathology is suggested by in vitro and in vivo
studies. Primary neuronal cultures expressing G2019S-LRRK2 develop *-synuclein
inclusions that can be reduced by LRRK2 inhibitor treatment. In vivo, infection
of a transgenic G2019S-LRRK2 rat model of Parkinson*s disease with a virus
overexpressing *-synuclein can induce dopaminergic neuron neurodegeneration,
and this degeneration can be attenuated by LRRK2 inhibitor treatment (Daher et
al. 2015; Volpicelli-Daley et al. 2016).
In summary, LRRK2 activity is linked to central mechanisms of Parkinson*s
disease pathology through its role in lysosomal function, and LRRK2 kinase
inhibitors, such as DNL201 (IND Number: 133665) represent a new class of
therapeutics with potential to address the underlying biology of Parkinson*s
disease.

In preparation for future clinical studies using LRRK2 kinase inhibitors
currently in development, the current study aims to characterize several
potential pharmacodynamic markers of LRRK2 kinase inhibition and patient
stratification. In particular, since LRRK2 is present in peripheral as well as
central compartments, the present study will examine these markers in the blood
and CSF compartments, and markers will be characterised in terms of both intra-
and inter- subject variability. Patients with Parkinson*s disease with LRRK2
mutations, patients with PD without LRRK2 mutations, and healthy matched
subjects will be studied to assess differences in biomarker-based phenotypes
between the groups, as well as intra-subject variability and inter-subject
variability within groups.

To investigate Day-to-day (intra-individual), and Inter-individual variability,
a design with repeated measurements over two days has been selected.
Participation comprises a total of up to 3-4 visits.

This study requires collection of skin, blood, CSF and urine samples. All
collections will be performed in a state of the art clinical unit and medically
supervised by qualified medical staff. The burden for the volunteer related to
the study procedures will be kept to a minimum.
Genotyping the LRRK2 and GBA genes is a mandatory part of study participation
if this has not already been done previously. Currently, confirmation of a
LRRK2 or GBA mutation has no implications for PD treatment. Healthy Volunteers
will undergo genotyping for Parkinson disease associated mutations in both
genes, but will not be informed of the outcome of the genotyping.
If a PD patient does not wish to know their LRRK2 or GBA status, they cannot
participate in the study.