Sphingolipidomics extraction and targeted omics from dried blood spots (DBS) to enable a study on MPS across three Canadian sites

A clinical trial is currently underway in the US to assess the effectiveness, safety, and
pharmacokinetics of a TNF-alpha inhibitor in treating inflammation and pain in MPS. We’re trying to expand the trial into Canadian sites, research indicates the role of sphingolipids as key inflammatory agents. Notably, TNF-alpha impacts inflammation by affecting sphingolipid signaling route. We successfully developed a reliable extraction method of two sphingolipids species, ceramides and sphingomyelins, using dried blood spot (DBS) collection for consistency and convenience, and an analytical method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for its specificity and sensitivity. The method validation process included linearity, recovery, reproducibility, precision and accuracy tests that have shown promising results within FDA guidelines, consequently achieving our research objective. This method therefore holds potential for clinical labs adoption because of its quickness and low margin of error. By incorporating this new screening method we could potentially increase the effectiveness of the researched pain reliever medication and eventually mark a significant step towards improving the quality of life for individuals with MPS and other diseases

DEVELOPING A TARGETED SPHINGOLIPIDOMICS METHOD TO QUANTIFY 1-DEOXYSPHINGOLIPIDS FROM PLASMA OF MUCOPOLYSACCHARIDOSES PATIENT

The primary aim of my summer research project is to establish a methodology for quantifying pro-inflammatory 1-deoxysphinganine and 1-deoxysphingosine levels in the bloodstream of individuals afflicted with Mucopolysaccharidoses (MPS). Our research has made substantial strides in the development of dependable quantification methods for 1-deoxysphinganine and 1-deoxysphingosine, illuminating their potential roles in MPS and related lysosomal storage diseases. These findings not only deepen our current comprehension of these conditions but also lay a sturdy groundwork for forthcoming inquiries, enabling us to establish a standardized approach for quantifying these molecules. This standardization will facilitate the adoption of this method in future studies and clinical laboratories.

Chaperone Therapy for MPSIIIC 

Glucosamine is an inhibitor of HGSNAT that showed promising signs of chaperone effect on the mutant misfolded enzyme in cultured skin fibroblasts of MPSIIIC patients4. Moreover, most recent results from Pshezhetsky lab show that glucosamine partially rescues misfolded mutant HGSNAT and ameliorates pathology on the mouse model of MPSIIIC, expressing HGSNAT with a human misfolding mutation P304L (HgsnatP304L strain). However, the doses of glucosamine used in the mouse study (2 g/kg BW per day) are not achievable in human patients. Therefore, we plan to produce and test derivatives of 2-2-deoxy-glucosamine (GlcNH2) that feature basic, labile, and bulky groups. The goal is to test if the active site of HGSNAT features a binding pocket that is optimized to complex the basic sidechain of its native substrate. These compounds have been already provided to us by our research partners from the University of Alberta. 

Cathepsin B As a Trigger of β-amyloid Deposition In Neurological Mucopolysaccharidoses

1. Rationale   

Recent study from the Pshezhetsky lab (11) demonstrated that in the mouse MPS I model, the deposition of amyloid plaques was associated with increased cathepsin B (CATB) activity leading to an alternative pro-amyloidogenic processing of the amyloid precursor protein (APP). Their unpublished data showed that similar pathway exists in the brain of MPS IIIC mice, suggesting a potential role of CATB in the neuropathology of MPS in general. 

1. Research plan 

In order to test the hypothesis that CATB is involved in deposition of amyloid plaques in MPS IIIC mice, the laboratory of Dr. Pshezhetsky generated a double-knockout mouse model (Hgsnat-/-CatB-/-) of MPS IIIC depleted on CATB activity. By themself, the CATB KO mice do not express any pathological phenotype. I will test whether CATB-deficient MPS IIIC mice show amelioration of the disease progression as compared with the original MPS IIIC mice. In particular, I will study their memory by Y-maze and Novel Object Recognition behavioral tests. I will also study the CNS pathology, including neuronal accumulation of Thioflavin-S-positive misfolded protein aggregates, deposition of amyloid plaques and levels of neuroinflammatory GFAP-positive astrocytes and CD11b-positive activated microglia in brain cortex by confocal fluorescent microscopy. Finally, I will directly analyze level of the 16-kDa C-terminal pathogenic APP fragment by Western blots. 

1. Significance

The proposed study should confirm existence of a novel CATB-associated alternative amyloidogenic pathway in MPS brain induced by lysosomal storage and potentially leading to neurodegeneration. It also may suggest a novel therapeutic approach for reduction of neurodegeneration and neuroinflammation in MPS by inhibiting CATB activity with small molecule drugs. 

Development and Validation of an Extraction Liquid Chromatography Tandem Mass Spectrometry Method To Evaluate the Role of Sphingolipid–1-Phosphate Species in MPSs

Objective and Specific Aims 

As part of our objective to develop methods for comprehensive analysis of sphingolipids in MPS, JeaYeon will develop and validate an extraction – liquid chromatography – tandem mass spectrometry technique to measure and analyse the family of sphingolipid-1-phosphate (C1P, ceramide-1-P and S1P, sphingosine-1-P) species in serum of MPS patients.

In a recent study by Simonaro et.al. (2008), treatment of healthy synovial fibroblasts with GAGs led to production of S1P and showed an increase in cell proliferation, consistent with the hyper plastic synovial tissue in MPS patients. Quebec has a founder effect for Morquio A disease (MPS IV). About 50 patients are followed at the MUHC by Dr John Mitchell, giving us a unique access to clinical samples.  

The main objective of this project will be achieved by addressing the following specific aims

Specific aim 1 (~4-5 weeks): 

To develop tandem mass spectrometry (multiple reaction monitoring) methods to analyse and measure sphingolipid-1-phosphate species on a triple quadrupole MS instrument.

Specific aim 2 (~2-3 weeks)

To develop extraction methods of sphingolipid-1-phosphate from serum and/or dried blood spots Specific aim 3 (~4 weeks) 

To measure the levels of sphingolipid-1-phosphate species in samples of MPS patients and healthy controls. Identify whether sphingolipid-1-phosphate can be used as a primary clinical biomarker or potential target for MPS disorders.

Inhibitory Potential of Class I SRT Candidates in Human Cells Including Neuronal-like Cells

We have identified NDST1 as druggable target for substrate reduction therapy (SRT) of mucopolysaccharidoses, specifically MPS type III (Sanfilippo disorder). 

In this project, we will use in vitro assays, to identify lead candidates for the treatment of patients with Sanfilippo disease.

Our class I (gene repressor) SRT candidates were identified by high-content screening of Hek293 cells with an inserted reporter gene. The efficacy of the candidates needs to be confirmed in other human cells including cells resembling human nervous tissues since the regulatory motives necessary for the control of NDST1 expression in these cells may be slightly different. Be2C is a human neuroblastoma cell line whose cell division is under direct control of retinoic acid. LUHMES cells are derived from embryonic human mesencephalon and immortalized by introducing a tetracycline-responsive v-myc gene. They can be easily cultured and maintained, like other cell lines. Both cell lines are well established in our research group. 

We will assess the ability of Class I SRT candidates to reduce NDST1 mRNA, protein and enzyme activity with qPCR, western blot and enzyme activity studies in HeLa, Hek293 cells, MPS III A/B/C, and in neuronal-like cell lines Be2C and LUHMES.

CRISPR Based Prime Editing For Correction of MPS Causative

We propose using base and prime editing for correction of causative mutations in MPS diseases. We have designed CRISPR-Cas9 base and prime editing constructs (guide RNA; gRNA) targeting reported patient mutations in ten MPS diseases1. gRNA and either base or prime editors will be delivered to HEK293T cells and analyzed for on-target editing. Editing constructs successful in HEK293T cells will then be tested in patient cells.

SLC26A11 Dysfunction in a New Mucopolysaccharide Turnover Disorder 

Preliminary  data: We  studied  a  patient  with  the  following  characteristics: consanguineous parents, microcephaly, intellectual disability, proptosis, hip dysplasia, coxa vara, lordosis, club feet, and significant joint hypermobility at birth. She was given a likely diagnosis of Larsen syndrome at birth, but  now  at  young  adult  age,  the  phenotype  resembles  more  Ehlers-Danlos  syndrome  ,  musculocontractural type.  A  mucopolysaccharide  biosynthesis  defect  was  suspected  on  clinical  grounds,  but  panel  sequencing  of known  MPS  biosynthesis  disease  genes  was  negative.  Exome  sequencing  revealed  a  homozygous  missense  in another  sulfate  transporter,  SLC26A11  (Y69C). The  mutated  residue  is  highly  conserved,  and  this  residue  was shown  to  be  critical  in  plants.3 This protein localizes at the lysosome, where it was proposed to serve as a sulfate exporter.4-6 We have obtained fibroblasts from this patient and stably corrected them with a lentivirus encoding wildtype SLC26A11, using methods we’ve published previously. 

Hypothesis  and  objective:    We  hypothesize  that  SLC26A11  dysfunction  leads,  in  cells  with  a  rapid  MPS turnover, to sulfate accumulation in lysosomes, and decreased cytoplasmic availability of sulfate essential for MPS synthesis.  Our objective is to demonstrate this hypothesis in patient samples and cells. 

Our project will allow us to confirm our hypothesis that SLC26A11 mutations cause lysosomal dysfunction and decreased MPS synthesis, and will also allow us to test a potentially new therapy.

Efficacy of Hematopoietic Stem/Progenitor Cell/LV Gene Transfer in the Mouse Model of Mucopolysaccharidosis III Type C. 

The  treatment  of  MPS  IIIC  is  currently  not  available,  however  emerging  data  have  shown  that  Lentivirus  (LV)-mediated  gene  correction  of  hematopoietic  stem/progenitor  cells  (HSPC)  followed  by  their  transplantation provides an efficient method to treat neurological LSDs. Following this idea, we proposed a research program aimed  at  treating  MPSIIIC  disease  by  the  supraphysiological  delivery  of  wild-type  (WT)  HGSNAT  to  the  brain microglia via transplantation of gene-modified HSPC. 

Last  summer,  we  tested  the  efficacy  of  LV-mediated  targeting  of  the  WT  HGSNAT  cDNA  into  mouse  HSPC  cell culture. We showed that LV vectors can be used for the transfer of wild-type HGSNAT gene to mouse HPSC for further  transplantation  into  MPSIIIC  mice.  Our  results  indicated  that  the  transduction  efficiency  of  the lentivirus  was  too  low  in  macrophage  culture,  we  had  to  modify  our  protocol  to  optimize  the  transduction efficiency  of  lentiviral  vectors.  We  found  that  using  two  hits  of  lentivirus  at  MOI  30  within  48  hours  yielded better amounts of transduced HSPC than a single hit at MOI 60. This approach was used in our pilot experiment where  we  transplanted  our  MPS  IIIC  mice  with  HSPC  transduced  with  the  lentivirus  encoding  HGSNAT-GFP enzyme. We found that the engraftment of donor cells in some transplanted MPS IIIC mice was as high as 80%. Furthermore,  these  mice  also  showed  improved  behavior  in  the  open  field  test  and  had  normalized  HGSNAT activity in the bone marrow and spleen, but not in the brain. 

The  main  objective  of  my  summer  research  project  is  to  improve  LV-mediated  targeting  of  the  WT  HGSNAT cDNA  into  the  brain  of  our  mouse  model  and  to  test  the  efficacy  of  this  lentivirus  by  carrying  out  in  vivo experiments  in  a  higher  number  of  MPS  IIIC  KO  mouse  model. 

Analysis Of Serum Cytokine Profile In Mucopolysaccharidoses

This summer research project aims at further characterising the mechanism of GAGs mediated lesions in MPS disorders by examining the levels of pro-inflammatory cytokine and chemokines in the serum of MPS patients.

At the laboratory of Dr John Mitchell, we have unique access to clinical samples from patients at the MUHC and other health centres in Canada. Specific cytokines and chemokines levels in serum of MPSs patients will be quantified using biochemical tools. We will evaluate the levels of TNF-α, IL-1β, IL-6, IL-8, IL-10, MCP-1, MIP-1a and MMP-9 using Quansys (Q-PlexTM) kits. The kit performance is optimised for analysis of precise proteins in human samples. The method provided by this kit is based on ELISA (enzyme-linked immunosorbent assay) concept.

Our ultimate aim is to probe the inter-relationship between serum inflammatory markers (cytokine), the serum level of ceramides (ceramides quantification is part of another project run in our laboratory), and the clinical phenotype.

Development And Validation of a Robust Dried Blood Spot Ceramide Profiling Method To Study Metabolism In Mucopolysaccharidoses

 Last year, we developed a sensitive targeted sphingolipidomics approach to evaluate the ceramide content in serum of MPS patients. The ceramide quantification method employs an electrospray tandem mass spectrometry approach coupled to reverse phase liquid chromatography. Interesting results have been obtained as per the levels of glucosylceramide and dihydroceramide species.

As a continuation of our last year’s project, the objective of this study is to translate the method of ceramide quantification in serum to a method allowing ceramide quantitation in dried blood spots (DBS). Ultimately, we aim to determine how abnormalities of the blood ceramide profile relate to the pathogenesis in MPS disorders, and whether ceramide could be used as a biomarker for diagnosis, follow up and potential therapeutic target.

Identification Of Plasma Sphingolipids Biomarkers For Morquio Type A

 The research study will be:

Part 1- To develop an extraction procedure (intra-source separation) to purify a specific category of sphingolipids from serum (e.g.: gangliosides, cerebrosides and lactosyl ceramide).  

Part 2- To develop initial profiling of various standards of sphingolipids to determine the appropriate MRM pairs used for Liquid Chromatography tandem Mass Spectrometry (LC MS/MS). 

Part 3- Ultimately, to quantitate specific plasma sphingolipid biomarkers by LC MS/MS, using internal standard corresponding to the biomarker quantified.

Mucopolysaccharidosis IIIB (MPS IIIB, Sanfilippo B Syndrome): Uptake Of Human Recombinant Naglu Utilizing PTD4-Mediated Transport In MPS IIIB Fibroblasts 

The research focus of the Choy lab is to investigate if PTD4, a synthetic derivative of Tat, will facilitate the delivery of recombinant Naglu across BBB thereby improving the bioavailability of recombinant Naglu in the CNS. Previous research in the Choy lab has fused Naglu to PTD4 and stably expressed this fusion construct in Spodoptera frugiperda (Sf9) cells to establish the economical, large-scale production Naglu-PTD4 (7,8). 

Populational MPS Neonatal Screening By MS/MS Using Urine Samples Collected On Filter Paper  

We are planning to adapt, optimize and validate the HPLC-MS/MS method using urine samples collected on filter paper to screen all newborns in the province of Quebec for MPSs as part of an evaluative research project.

Different technological steps are involved: 1) Optimize the GAG extraction procedure from the filter paper samples;

2) Develop a robust and rapid ultra-performance liquid chromatography (UPLC) method that allows an adequate separation of HS, DS, CS and creatinine;

3) Optimize a quantitation method using tandem mass spectrometry (MS/MS);

4) Perform a complete validation of the method to ensure that it is robust for a newborn mass screening research project.

Following the validation of the method, we will analyse urine samples collected on filter paper from 21-day old babies to establish reference control values.  

Ultimately, we plan to add this new method to the already existing neonatal urine screening program in the province of Quebec as part of an evaluative research endeavour.

Our aim is to detect newborns with MPS as early as possible, and to provide a rapid intervention before the onset of symptoms.