Project

Fix Neutropenia (FIXNET): focusing on neutrophil proteases defects which serve as novel diagnostic and therapeutic options.

Childhood neutropenia is a heterogeneous group of immunodeficiency and is characterized by a significantly reduced count of neutrophilic granulocyte. Neutrophils as the most abundant type of white blood cells are an essential part of the innate immune system, they ingest, kill, and digest microbial pathogens.

The current neutropenia diagnosis is based on serial blood morphology test and clinical features such as recurrent infections and mucositis. Distinguishing the molecular basis of the diseases it is not possible at the diagnosis, thus doctors don’t know whether the patient suffers from congenital neutropenia or transient form, resulted from immunological reason e.g. after a viral infection. In that case, the pediatrician ought to postpone protective vaccinations, which leads to over 3,000 cases of unvaccinated children annually in Poland. In congenital neutropenia with genetic defects, there is a need to develop a faster and cheaper method of comprehensive diagnosis and treatment. Nowadays, the mutation of the gene responsible for this disease is unknown in about 30-40% of congenital neutropenia patients.

The main cause of neutrophils deficiency are inhibition of granulocytes maturation, arrest in bone marrow, or molecular defects inducing apoptosis. Neutrophils contain specialized granules that possess many proteins, including specific neutrophilic serine proteases. These proteases are necessary for the maturation and functioning of the entire cell, thus mutations in proteases encoding genes lead to neutrophil defects.

The project involves combining the clinical and genetic data with the biology of neutrophilic serine proteases. The main goals are the search for defects in new genes that cause neutropenia, identification of the protease role in neutrophil disorders, designing a unique diagnostic test, and an effective and safe targeted therapy method based on gene editing.

Stage 1

The search for patients with neutropenia for the evaluation of their clinical characteristics.

  • The recruitment of patients with immune-related neutropenia, congenital neutropenia and their family’s members.
  • Collection of the blood, urine and bone marrow samples from recruited patients with various cause of neutropenia.

time-frame: 1-18 month of the project

Stage 2

The targeted genetic analysis of the patients with congenital neutropenia.

  • Identification of the genetic cause within known genes associated with neutropenia.

time-frame: 6-24 month of the project
methods: NGS sequencing

Stage 3

The whole exome/whole genome genetic analysis of the patients with congenital neutropenia.

  • Identification of the putative genetic cause associated with neutropenia.

time-frame: 18-48 month of the project
methods: NGS sequencing

Stage 4

The generation of the iPS cells repository from the patients with congenital neutropenia.

  • Preparation of the iPS cells from patients with various identified mutation causing neutropenia.

time-frame: 3-18 month of the project
methods: iPS generation

Stage 5

Gene editing for correction of the various identified mutation causing neutropenia in human cells.

  • The cell correction using CRISP/CAS9 gene editing from patients with various identified mutation causing neutropenia and preparing these cells for functional studies.

time-frame: 6-36 month of the project
methods: CRISPR/Cas9 gene editing technology

Stage 6

The application of fluorescent-labelled activity-based probes to dissect the distribution of active neutrophil serine proteases (NSPs) within particular organelles in neutrophils.

  • Creation of a first subcellular map of the activity of NSPs in neutrophils from healthy donors and from neutropenia patients with the fluorescence microscopy.

time-frame: 1-48 month of the project
methods: HyCoSuL, confocal microscopy

Stage 7

The synthesis and application of metal-labelled activity-based probes to dissect the activity of NSPs and their interactions with serpins in neutrophils from healthy donors and neutropenia patients.

  • Development of a chemical procedure for the high yield and high purity synthesis of NSPs activity-based probes tagged with stable isotopes of metals.
  • Development of an integrated chemical biology platform, dedicated to the investigation of neutrophils, which will consist of (1) metal-tagged chemical probes for active NSPs, (2) metal-tagged antibodies for total NSPs, and (3) metal-tagged antibodies for NSPs`endogenous inhibitors.

time-frame: 1-48 month of the project
methods: mass cytometry

Stage 8

Functional analysis of samples from patients suffering from neutropenia and healthy individuals.

  • Functional neutrophil studies with the defects observed in at least 80 individuals.

time-frame: 3-42 month of the project
methods: functional neutrophil studies

Stage 9

Generation of a mouse model with mutations within selected genes.

  • Preparation of the mice models with the defects similar to observed in patients with various identified mutation causing neutropenia.

time-frame: 3-30 month of the project
methods: CRISPR/Cas9 gene editing technology

Stage 10

Verification of neutrophil functionality in infectious mouse models in vivo.

  • Functional neutrophil studies from the mice models with the defects similar to observed in patients with various identified mutation causing neutropenia.

time-frame: 12-48 month of the project
methods: mouse infection model

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