15th European Molecular Imaging Meeting
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Young Investigator Award

Session chair: Giannis Zacharakis (Heraklion, Greece); Frauke Alves (Göttingen, Germany)
 
Shortcut: PL 08-10
Date: Friday, 28 August, 2020, 9:00 a.m.
Session type: Plenary Session

Contents

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1:30 p.m. PL 08-10-01 *.ics

In vivo PET imaging of mutant huntingtin using [11C]CHDI-180R as candidate marker in a mouse model of Huntington’s Disease

Daniele Bertoglio1, Jeroen Verhaeghe1, Klaudia Cybulska1, 2, Špela Korat1, 2, Alan Miranda1, Leonie Wyffels1, 2, Sigrid Stroobants1, 2, Vinod Khetarpal3, Ladislav Mrzljak3, Celia Dominguez3, Jonathan Bard3, Mette Skinbjerg3, Longbin Liu3, Ignacio Munoz-Sanjuan3, Steven Staelens1

1 University of Antwerp, Molecular Imaging Center Antwerp (MICA), Wilrijk, Belgium
2 Antwerp University Hospital, Department of Nuclear Medicine, Edegem, Belgium
3 CHDI Management/CHDI Foundation, Los Angeles, United States of America

Introduction

Huntington’s Disease (HD) is a progressive autosomal dominant neurodegenerative disorder caused by mutant huntingtin (mHTT). Although several promising therapeutic approaches aimed at decreasing cerebral mHTT levels are currently being developed, there is a major (pre)clinical need for a noninvasive marker to monitor mHTT changes. The aim of this study was to investigate the first in class mHTT PET radioligand, namely [11C]CHDI-180R, for imaging of mHTT aggregates in the Q175DN mouse model of HD.

Methods

Dynamic microPET/CT imaging was performed longitudinally in heterozygous (HET) Q175DN mice (n = 23) and wild-type (WT) littermates (n = 20) at 3, 6, 9, and 13 months of age. Total volume of distribution (VT) (Logan) was calculated noninvasively using an image-derived input function (IDIF) (VT (IDIF)) for brain regional and voxel-wise analyses. Post-mortem autoradiography with [3H]CHDI-180 and mHTT immunohistochemistry were performed at each timepoint (n = 10/genotype at 3, 6, 9, and 13 months of age) to confirm the in vivo findings. Receiver operating characteristic (ROC) curves for assessment of diagnostic ability and sample size calculations at desired therapeutic effects were performed.

Results/Discussion

Longitudinal PET imaging could discriminate HET from WT littermates as early as 3 months of age (striatum: HET = 0.53±0.03 mL/cm3, WT = 0.49±0.03 mL/cm3; +6.5%, p<0.001). Binding in HET mice increased successively over age, resulting in the largest difference between genotypes at 13 months of age (striatum: HET = 0.87±0.05 mL/cm3, WT = 0.48±0.03 mL/cm3; +82.5%, p<0.0001) (Fig. 1). Post-mortem analyses supported the in vivo PET quantification demonstrating increased binding with age in HET mice, which was associated with disease progression (r>0.89, p<0.0001) as measured by in vitro [3H]CHDI-180 autoradiography and mHTT immunohistochemistry. No age-related or disease progressive increase in PET signal was detectable in WT littermates. ROC curves revealed good accuracy already at 3 months of age (AUC = 0.81) (Fig. 2A). Sample size calculations shown that less than 10 animals per treatment group were required to detect a 30% therapeutic effect as of 6 months of age (Fig. 2B).

Conclusions

We described the first radioligand to image mHTT in the living brain. These findings indicate [11C]CHDI-180R PET imaging is a non-invasive candidate PET tracer suitable for monitoring HD progression and potentially for evaluating the efficacy of mHTT lowering therapies with future clinical application.

Longitudinal [11C]CHDI-180R PET imaging of mHTT
A Average VT (IDIF) parametric maps of [11C]CHDI-180R in WT and HET Q175DN mice at 3, 6, 9, and 13 months of age. B Regional [11C]CHDI-180R VT (IDIF) quantification in HET Q175DN mice and WT littermates. ***p<0.001, ****p<0.0001.
Evaluation of [11C]CHDI-180R as a candidate marker for disease-modifying therapies
A ROC curves for the diagnostic capacity between WT and HET mice at different stages of disease progression. WT: n = 12-19; HET: n = 17-23. AUC = area under the curve. B Sample size calculations at desired therapeutic effects for the design of a disease-modifying interventions using [11C]CHDI-180R PET imaging as endpoint.
Keywords: mHTT, biomarker, Huntington's Disease, Animal model, PET imaging
1:45 p.m. PL 08-10-02 *.ics

Imaging In Vivo CRISPR/Cas9 Neuronal Gene Editing in the Adult Rat Brain

Sabina Marciano1, Andreas Maurer1, Deniz Kirik2, Bernd J. Pichler1, Kristina Herfert1

1 University of Tuebingen, Werner Siemens Imaging Center, Department of Preclinical Imaging and Radiopharmacy, Tuebingen, Germany
2 Lund University, BRAINS Unit, Department of Experimental Medical Science, Lund, Sweden

Introduction

Gene editing in the adult rat brain, using CRISPR/Cas9 to interrogate gene function, has been hampered by the size of the CRISPR-associated endonuclease SpCas9 and the low in vivo efficiency of other Cas9 homologs. Here, we present a highly efficient gene editing approach, using Cas9 from Staphylococcus aureus (SaCas9) to target the Slc18a2 gene, encoding the vesicular monoamine transporter 2 (VMAT2), in vitro and in vivo. We use in vivo positron emission tomography (PET) imaging to investigate molecular changes at pre- and postsynaptic terminals and behavioural tests to explore the phenotype.

Methods

We first selected the most efficient sgRNA targeting the Slc18a2 in vitro in rat primary neurons, using adeno-associated viral (AAV) vectors, and validated the knockdown (KD) efficiency by surveyor assay and immunofluorescence. Next, AAV-SaCas9 and AAV-sgRNA targeting Slc18a2 (n=14) or LacZ (control, n=10) were stereotactically injected into the right substantia nigra of adult rats. Starting 8 weeks after AAVs delivery, VMAT2 expression, DAT availability, postsynaptic changes and inflammatory responses, were quantified using dynamic PET imaging with [11C]DTBZ, [11C]methylphenidate (MP), [11C]raclopride (RAC) and the TSPO ligand [18F]GE-180, respectively. CRISPR-induced VMAT2 KD was further characterized with behavioral readouts of locomotor activity, gait, spontaneous and evoked rotations.

Results/Discussion

We observed a -60/+2% [11C]DTBZ decrease indicative for VMAT2 KD in the right striatum compared to the left striatum (p=0.0001). The KD did not affect [11C]MP or [18F]GE-180 binding indicating that VMAT2 expression was selectively reduced without inducing neuronal loss or inflammation. As the reduction of dopamine (DA) at presynaptic terminals is known to result in postsynaptic changes of DA receptors, we performed [11C]RAC scans and observed a 3/40% increase in the right striatum compared to the left striatum (p=0.0003). Remarkably, changes in [11C]DTBZ binding strongly correlated to changes in [11C]RAC binding (R2=0.77). We did not observe changes for the tested radioligands in the LacZ-injected group. Additionally, VMAT2 KD rats revealed significant motor alterations, partly correlating with [11C]DTBZ binding changes, in terms of paw use preference (p=0.002, R2=0.72), total distance travelled (p=0.03), gait (p=0.01), spontaneous rotation (p=0.03, R2=0.20), evoked rotation (p=0.03).

Conclusions

Our work pioneers the combinatorial use of CRISPR/Cas9 gene editing and PET imaging to inspect in vivo the direct link between specific genes and molecular changes observed in pathology. We used CRISPR/Cas9 to edit the Slc18a2 in the adult rat brain, reproducing motor symptoms of Parkinson´s disease. We observed an effective DA depletion in the absence of neuronal loss, which strongly correlates with DA receptors occupancy or expression changes.

Keywords: PET, CRISPR/Cas9, Dopamine, Dopamine receptors expression changes, Parkinson`s disease
2:00 p.m. PL 08-10-03 *.ics

Assessing tumour cell death in vivo using 2H-labeled fumarate and deuterium magnetic resonance spectroscopic imaging

Friederike Hesse1, Vencel Somai1, 2, Felix Kreis1, Flaviu Bulat1, 4, Kevin Brindle1, 3

1 University of Cambridge, Cancer Research UK Cambridge Institute, Cambridge, United Kingdom
2 University of Cambridge, Department of Radiology, Cambridge, United Kingdom
3 University of Cambridge, Department of Biochemistry, Cambridge, United Kingdom
4 University of Cambridge, Department of Chemistry, Cambridge, United Kingdom

Introduction

Cell death is an important imaging target for assessing early tumour treatment response and the effectiveness of therapy. The degree of tumour cell death can be a predictive indicator of patient outcome. Traditionally, imaging modalities available in the clinic have focused on detecting late changes in tumour size rather than evaluating early changes in tumour physiology or metabolism. Here we investigated whether fast deuterium MRI can be used to detect cell death and assess early tumour treatment response using a new imaging biomarker, 2H-fumarate.1

Methods

EL4 lymphoma-bearing mice were imaged using a fast 3D deuterium MRI pulse sequence with a time resolution of 5 minutes, and a spatial resolution of 3 x 3 x 9 mm, following a bolus injection of [2,3-2H2] fumarate, before and 48 h after treatment with a chemotherapeutic drug (etoposide at 67 mg/kg). Experiments on EL4 cells in vitro were conducted on a 14.1 T high-resolution NMR spectrometer and experiments on tumours in vivo using a 7.0 T horizontal bore magnet.

Results/Discussion

Results:
As early as one minute after 2H-fumarate addition, etoposide-treated EL4 cells showed malate production, which was greater than that in untreated controls (Figure 1). A fast, chemical shift imaging sequence was used to assess conversion of fumarate to malate in implanted EL4 tumours in vivo following a bolus injection of labelled fumarate (1g/kg) to tumour-bearing mice. Within 48 h of drug treatment the malate/fumarate ratio increased significantly from 0.034 ±0.06 to 0.33 ±0.11 (p=0.04, n=3).
Discussion:
Deuterium metabolic imaging (DMI) with fumarate has great potential for quantitative assessment of tumour cell death in vivo. Compared to hyperpolarized 13C-labeled substrates, 2H-labeled metabolites are relatively easy to synthesize and at lower cost. However, image acquisition times are longer, and resolution is lower. Nevertheless, the first 2H MR experiments in a clinical setting have been conducted recently following oral administration of glucose.2

Conclusions

Tumour malate production from [2,3-2H2]fumarate increased significantly within 48 h of drug treatment, demonstrating the potential of DMI for assessing tumour cell death and the early responses of tumours to treatment.

References
[1] Gallagher, F. A. et al. 2009, Production of hyperpolarized [1,4-13C2]malate from [1,4-13C2]fumarate is a marker of cell necrosis and treatment response in tumors. Proc. Natl. Acad. Sci.106, 19801–19806
[2] De Feyter, H. M. et al. 2018, Deuterium metabolic imaging (DMI) for MRI-based 3D mapping of metabolism in vivo. Sci. Adv.4, eaat7314
2H NMR spectra of murine lymphoma (EL4) cell culture medium

Figure 1: (a-d) 2H NMR spectra of murine lymphoma (EL4) cell culture medium. a) Medium from untreated cells and b) cells treated for 24 h with etoposide, 3 minutes after the addition of fumarate. c-d) Medium from untreated cells and b) cells treated for 24 h with etoposide, 24 h after the addition of fumarate. e-f) Deuterated fumarate, malate and water concentrations in e) medium from etoposide-treated and f) untreated cells. g) Malate production in untreated and etoposide-treated EL4 cell suspensions (n=3 biological replicates). Error bars show the standard error.

Metabolite concentration maps derived from summed 3D CSI images

Figure 2: Metabolite concentration maps derived from summed 3D CSI images acquired over a period of 60 min following [2,3-2H2]fumarate injection into EL4 tumor-bearing mice. The color code represents concentration (in mM) derived from the ratios of the peak amplitudes in the malate and fumarate maps to peak amplitudes in an initial HDO map and corrected for the number of 2H labels per molecule and for signal saturation. Concentration maps of a) fumarate pre-treatment; b) malate pre-treatment; c) fumarate 48 h post-treatment; d) malate 48 h post-treatment.

Keywords: Deuterium metabolic imaging, 2H fumarate, metabolic imaging, Imaging tumour cell death