Podcast
Questions and Answers
What is the term for the process of generating mature blood cell components?
What is the term for the process of generating mature blood cell components?
- Phagocytosis
- Homeostasis
- Mitosis
- Hemopoiesis (correct)
Which of the following is a mature cellular component produced during hemopoiesis?
Which of the following is a mature cellular component produced during hemopoiesis?
- Platelets (correct)
- Muscle cells
- Nerve cells
- Epithelial cells
Which mature blood cell lacks a nucleus?
Which mature blood cell lacks a nucleus?
- White blood cells (WBCs)
- Red blood cells (RBCs) (correct)
- Lymphocytes
- Platelets
Granulocytes are a type of:
Granulocytes are a type of:
Which of the following are granulocytes classified into?
Which of the following are granulocytes classified into?
Platelets are fragments derived from what type of cell?
Platelets are fragments derived from what type of cell?
What is the approximate lifespan of red blood cells (RBCs)?
What is the approximate lifespan of red blood cells (RBCs)?
In adults, hematopoiesis primarily occurs in which location?
In adults, hematopoiesis primarily occurs in which location?
Hematopoietic stem cells initially emerge from which location during embryonic development?
Hematopoietic stem cells initially emerge from which location during embryonic development?
Which site has a high concentration of hematopoietic stem cells that are normally discarded after birth?
Which site has a high concentration of hematopoietic stem cells that are normally discarded after birth?
In adults, what percentage of bone marrow is expected to be present in a normal sample?
In adults, what percentage of bone marrow is expected to be present in a normal sample?
Hematopoiesis follows a hierarchical tree model, with which cells at the top?
Hematopoiesis follows a hierarchical tree model, with which cells at the top?
What is the name of the first marker cells express?
What is the name of the first marker cells express?
What is a key characteristic of hematopoietic stem cells (HSCs)?
What is a key characteristic of hematopoietic stem cells (HSCs)?
Which of the following best describes HSCs?
Which of the following best describes HSCs?
What are two specific laboratory findings that characterize aplastic anemia?
What are two specific laboratory findings that characterize aplastic anemia?
In aplastic anemia, a low reticulocyte count indicates which condition?
In aplastic anemia, a low reticulocyte count indicates which condition?
Why is there neutropenia and not lymphopenia in aplastic anemia?
Why is there neutropenia and not lymphopenia in aplastic anemia?
Aberrant autoreactive T lymphocytes in aplastic anemia target what?
Aberrant autoreactive T lymphocytes in aplastic anemia target what?
Which condition is NOT an exclusion from the definition of aplastic anemia?
Which condition is NOT an exclusion from the definition of aplastic anemia?
Trilinear cytopenia is a possible consequence of what condition?
Trilinear cytopenia is a possible consequence of what condition?
Pure red blood cell aplasia is an acquired autoimmune condition frequently associated with what?
Pure red blood cell aplasia is an acquired autoimmune condition frequently associated with what?
The myelodysplastic syndromes are classified as what kinds of diseases?
The myelodysplastic syndromes are classified as what kinds of diseases?
What is the most common etiology of aplastic anemia?
What is the most common etiology of aplastic anemia?
What characterizes hereditary aplastic anemia conditions?
What characterizes hereditary aplastic anemia conditions?
Fanconi anemia is characterized by alterations in genes involved in what process?
Fanconi anemia is characterized by alterations in genes involved in what process?
What is the most common technique for gene therapy, where cells are harvested and modified in the lab?
What is the most common technique for gene therapy, where cells are harvested and modified in the lab?
What is a common sign or symptom of aplastic anemia related to low platelet count?
What is a common sign or symptom of aplastic anemia related to low platelet count?
What finding characterizes aplastic anemia in the bone marrow?
What finding characterizes aplastic anemia in the bone marrow?
In the context of treating aplastic anemia, what factor is NOT typically considered when deciding between immunosuppression and allogenic hematopoietic stem cell transplant?
In the context of treating aplastic anemia, what factor is NOT typically considered when deciding between immunosuppression and allogenic hematopoietic stem cell transplant?
What cells are used as a therapy in CAR-T cell treatment?
What cells are used as a therapy in CAR-T cell treatment?
What is the purpose of introducing a new gene into T-cells during CAR-T cell therapy?
What is the purpose of introducing a new gene into T-cells during CAR-T cell therapy?
In allotransplantation, cells are taken from?
In allotransplantation, cells are taken from?
What is a primary target of CAR-T cell therapy?
What is a primary target of CAR-T cell therapy?
In what decade was the first transplant performed from a donor to a patient with acute leukemia?
In what decade was the first transplant performed from a donor to a patient with acute leukemia?
The first transplant in Europe was performed for what type of accident?
The first transplant in Europe was performed for what type of accident?
What was a major cause of patient deaths during the first transplant experiences in the late 1950s?
What was a major cause of patient deaths during the first transplant experiences in the late 1950s?
What is the main cause of major complications in transplantations?
What is the main cause of major complications in transplantations?
What is the best way to avoid graft-versus-host disease (GvHD)?
What is the best way to avoid graft-versus-host disease (GvHD)?
What is the term used to describe the immune reaction of the donor's immune system against the host?
What is the term used to describe the immune reaction of the donor's immune system against the host?
What is a significant downside to T-cell depleted allotransplantation??
What is a significant downside to T-cell depleted allotransplantation??
What does HSCT stand for?
What does HSCT stand for?
What must an antigen do to be recognized by CAR-T cells?
What must an antigen do to be recognized by CAR-T cells?
Where does the variable region of CAR-T cells, which recognizes the antigen, come from?
Where does the variable region of CAR-T cells, which recognizes the antigen, come from?
What does CD19 refer to in the context of CD19 CAR-T cell therapy?
What does CD19 refer to in the context of CD19 CAR-T cell therapy?
What are the six approved CAR-T cell therapies directed against?
What are the six approved CAR-T cell therapies directed against?
What characterizes the challenges related to failure in CAR-T cell therapy?
What characterizes the challenges related to failure in CAR-T cell therapy?
What is a common early side effect that occurs a couple of days after CAR-T cell infusion?
What is a common early side effect that occurs a couple of days after CAR-T cell infusion?
What is the first step in treating any toxicities resulting from CAR-T cell therapy?
What is the first step in treating any toxicities resulting from CAR-T cell therapy?
What term would be most appropriate to describe the nature of side effects to CAR-T cell treatment neurotoxicity?
What term would be most appropriate to describe the nature of side effects to CAR-T cell treatment neurotoxicity?
First-level analysis in hematology includes which of the following?
First-level analysis in hematology includes which of the following?
Which characteristic describes a complete blood count (CBC)?
Which characteristic describes a complete blood count (CBC)?
Morphology in first-level analysis is performed when?
Morphology in first-level analysis is performed when?
In first-level assays, what is the primary goal?
In first-level assays, what is the primary goal?
Which of the following is an example of a second-level analysis?
Which of the following is an example of a second-level analysis?
Where should blood draws be performed in order to perform CBCs, morphology, or flow cytometry?
Where should blood draws be performed in order to perform CBCs, morphology, or flow cytometry?
What's crucial to avoid when a blood sample is obtained?
What's crucial to avoid when a blood sample is obtained?
Why are anticoagulants like EDTA used when collecting blood samples?
Why are anticoagulants like EDTA used when collecting blood samples?
Which of the following anticoagulants is typically preferred because it doesn't affect cell morphology?
Which of the following anticoagulants is typically preferred because it doesn't affect cell morphology?
In first-level analysis, manipulation of the blood sample should be...
In first-level analysis, manipulation of the blood sample should be...
What type of stain is used upon finding alarming signals from a complete blood count?
What type of stain is used upon finding alarming signals from a complete blood count?
What does flow cytometry frequently analyze?
What does flow cytometry frequently analyze?
What is essential for interpreting flow cytometry results?
What is essential for interpreting flow cytometry results?
What does a complete blood count (CBC) generally count and characterize?
What does a complete blood count (CBC) generally count and characterize?
What is measured by the hematocrit?
What is measured by the hematocrit?
What does CBCs generally give information about?
What does CBCs generally give information about?
What is the concentration of hemoglobin in the blood expressed in?
What is the concentration of hemoglobin in the blood expressed in?
What does the blood mirror for the patient?
What does the blood mirror for the patient?
Anemia is a condition with which of the following?
Anemia is a condition with which of the following?
What cells are produced in the bone marrow?
What cells are produced in the bone marrow?
What do the asterisks on a CBC indicate?
What do the asterisks on a CBC indicate?
What is the normal range for MCV's in women?
What is the normal range for MCV's in women?
MCV is used to define anemia as:
MCV is used to define anemia as:
In myeloproliferative neoplasms, genetic alterations typically occur in which cells?
In myeloproliferative neoplasms, genetic alterations typically occur in which cells?
Unlike acute myeloid leukemia (AML), cells in myeloproliferative disorders:
Unlike acute myeloid leukemia (AML), cells in myeloproliferative disorders:
Which historical figure contributed significantly to the understanding of myeloproliferative syndromes?
Which historical figure contributed significantly to the understanding of myeloproliferative syndromes?
Chronic Myeloid Leukemia (CML) is characterized as a:
Chronic Myeloid Leukemia (CML) is characterized as a:
Polycythemia Vera (PV) is mainly characterized by:
Polycythemia Vera (PV) is mainly characterized by:
Essential Thrombocytosis (ET) is defined by an increase in:
Essential Thrombocytosis (ET) is defined by an increase in:
Primary Myelofibrosis (PM) is characterized by:
Primary Myelofibrosis (PM) is characterized by:
What is a common characteristic of myeloproliferative neoplasms, relating to organ size?
What is a common characteristic of myeloproliferative neoplasms, relating to organ size?
The growth rate of neoplastic cells in myeloproliferative neoplasms can be described as:
The growth rate of neoplastic cells in myeloproliferative neoplasms can be described as:
Many myeloproliferative neoplasms are often diagnosed when:
Many myeloproliferative neoplasms are often diagnosed when:
What organs are commonly infiltrated by leukemia in myeloproliferative diseases?
What organs are commonly infiltrated by leukemia in myeloproliferative diseases?
Polycythemia Vera and Essential Thrombocytosis are described as the most ______ of the myeloproliferative neoplasms.
Polycythemia Vera and Essential Thrombocytosis are described as the most ______ of the myeloproliferative neoplasms.
What is a potential risk of evolution in all myeloproliferative neoplasms?
What is a potential risk of evolution in all myeloproliferative neoplasms?
The observation of transformation into AML in myeloproliferative neoplasms has informed researchers about:
The observation of transformation into AML in myeloproliferative neoplasms has informed researchers about:
According to the WHO classification, myeloproliferative neoplasms are classified based on:
According to the WHO classification, myeloproliferative neoplasms are classified based on:
In addition to clinical symptoms and lab results, what other criteria are used to classify and understand myeloproliferative neoplasms?
In addition to clinical symptoms and lab results, what other criteria are used to classify and understand myeloproliferative neoplasms?
What diagnostic procedure is typically performed when suspecting a myeloproliferative disorder to quantity bone marrow?
What diagnostic procedure is typically performed when suspecting a myeloproliferative disorder to quantity bone marrow?
What is the molecular counterpart of the Philadelphia chromosome?
What is the molecular counterpart of the Philadelphia chromosome?
A mutation in the JAK2 gene, specifically in position 617, results in:
A mutation in the JAK2 gene, specifically in position 617, results in:
The Bcr-Abl translocation leads to the production of a:
The Bcr-Abl translocation leads to the production of a:
The Bcr-Abl translocation results in a tyrosine kinase that is:
The Bcr-Abl translocation results in a tyrosine kinase that is:
What does TKI stand for in the genetics of CML?
What does TKI stand for in the genetics of CML?
The tyrosine kinase produced in CML will phosphorylate adhesion molecules of the ________ proteins?
The tyrosine kinase produced in CML will phosphorylate adhesion molecules of the ________ proteins?
In CML, the classical mutation of Bcr-Abl leads to a protein of:
In CML, the classical mutation of Bcr-Abl leads to a protein of:
The natural history of CML is made of how many phases?
The natural history of CML is made of how many phases?
Which phase of CML is usually how the disease is diagnosed?
Which phase of CML is usually how the disease is diagnosed?
Symptoms of CML that arise from the accumulation of the disease include:
Symptoms of CML that arise from the accumulation of the disease include:
In CML, a complete blood count (CBC) will show:
In CML, a complete blood count (CBC) will show:
The Philadelphia chromosome is found using cytogenetics, and can be found in both:
The Philadelphia chromosome is found using cytogenetics, and can be found in both:
What additional test can be done after cytogenetic analysis?
What additional test can be done after cytogenetic analysis?
What is the primary focus of current gene therapy research related to hematopoietic stem cell transplantation?
What is the primary focus of current gene therapy research related to hematopoietic stem cell transplantation?
What does 'ex vivo' gene therapy involve?
What does 'ex vivo' gene therapy involve?
When introducing a CAR gene into T lymphocytes, what is the aim of this gene addition?
When introducing a CAR gene into T lymphocytes, what is the aim of this gene addition?
What is the main goal of gene subtraction in gene therapy?
What is the main goal of gene subtraction in gene therapy?
Which method is MOST associated with gene correction?
Which method is MOST associated with gene correction?
What is the primary difference between gene transfer and gene therapy?
What is the primary difference between gene transfer and gene therapy?
Why is the ability to integrate a gene into the recipient cell considered a significant milestone in gene therapy?
Why is the ability to integrate a gene into the recipient cell considered a significant milestone in gene therapy?
Which type of vector is MOST commonly used for integrating genes in hematopoietic stem cell-based gene therapy?
Which type of vector is MOST commonly used for integrating genes in hematopoietic stem cell-based gene therapy?
What is a key advantage of lentiviral vectors compared to gamma retroviral vectors?
What is a key advantage of lentiviral vectors compared to gamma retroviral vectors?
What is a defining characteristic of non-viral methods for gene transfer, such as transposons?
What is a defining characteristic of non-viral methods for gene transfer, such as transposons?
What is a significant limitation of RNA when used in gene therapy?
What is a significant limitation of RNA when used in gene therapy?
What dictates the area of the genome where guide RNAs bind in the CRISPR-Cas9 system?
What dictates the area of the genome where guide RNAs bind in the CRISPR-Cas9 system?
After a DNA double-strand break is created during genome editing, what is a 'quick and dirty' repair mechanism cells can use?
After a DNA double-strand break is created during genome editing, what is a 'quick and dirty' repair mechanism cells can use?
Which of the following is used as a template to repair DNA after a double-strand break using homologous directed repair (HDR)?
Which of the following is used as a template to repair DNA after a double-strand break using homologous directed repair (HDR)?
What is the primary advantage of CRISPR-Cas9 compared to older genome editing tools like ZFNs and TALENs?
What is the primary advantage of CRISPR-Cas9 compared to older genome editing tools like ZFNs and TALENs?
What is the purpose of the Cas protein nickase in base editing?
What is the purpose of the Cas protein nickase in base editing?
Why are hematopoietic stem cells (HSCs) frequently used in ex vivo gene therapy?
Why are hematopoietic stem cells (HSCs) frequently used in ex vivo gene therapy?
What is a key reason for targeting the lung and liver when designing vectors for IV injection in gene therapy?
What is a key reason for targeting the lung and liver when designing vectors for IV injection in gene therapy?
What is the overall survival rate since 2000 for patients with ADA-SCID treated with gene therapy?
What is the overall survival rate since 2000 for patients with ADA-SCID treated with gene therapy?
Gene therapy has improved for lysosomal disorders because modified enzymes are able to do what?
Gene therapy has improved for lysosomal disorders because modified enzymes are able to do what?
Flashcards
Hemopoiesis
Hemopoiesis
The process of generating mature cellular blood components.
Mature Cellular Components of Blood
Mature Cellular Components of Blood
RBCs, WBCs, and platelets
Primary Site of Hematopoiesis
Primary Site of Hematopoiesis
Bone marrow.
Nature of Hematopoietic Diseases
Nature of Hematopoietic Diseases
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Embryonic Development of Hematopoiesis
Embryonic Development of Hematopoiesis
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AGM
AGM
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Characteristics of a Stem Cell
Characteristics of a Stem Cell
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First Marker Expressed by HSCs
First Marker Expressed by HSCs
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Stem Cell Traits
Stem Cell Traits
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Two Main Niches of Hematopoietic Stem Cells
Two Main Niches of Hematopoietic Stem Cells
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Unique Growth Factor in Hemopoiesis
Unique Growth Factor in Hemopoiesis
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Facilitator of Granulocyte Generation
Facilitator of Granulocyte Generation
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Negative Regulators of Hematopoiesis
Negative Regulators of Hematopoiesis
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Harvesting HSCs
Harvesting HSCs
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Hematology and Oncology Diagnostics
Hematology and Oncology Diagnostics
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First-Level Analysis
First-Level Analysis
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Complete Blood Count (CBC)
Complete Blood Count (CBC)
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Morphology (blood smear)
Morphology (blood smear)
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Goal of First-Level Assay
Goal of First-Level Assay
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Second-Level Analysis
Second-Level Analysis
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Blood Sample
Blood Sample
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Avoiding Clotting
Avoiding Clotting
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EDTA
EDTA
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May Grunwald Giemsa staining
May Grunwald Giemsa staining
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Flow Cytometry Preparation
Flow Cytometry Preparation
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Need for Clinical Data in Flow Cytometry
Need for Clinical Data in Flow Cytometry
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Three Types of Blood Cells
Three Types of Blood Cells
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CBC Parameters
CBC Parameters
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Anemia
Anemia
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Circulating Blood Cells
Circulating Blood Cells
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MCV (Mean Corpuscular Volume)
MCV (Mean Corpuscular Volume)
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Describing Anemia
Describing Anemia
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Microcytic Anemia Causes
Microcytic Anemia Causes
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Macrocytic Anemia Causes
Macrocytic Anemia Causes
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MCH (Mean Corpuscular Hemoglobin)
MCH (Mean Corpuscular Hemoglobin)
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MCHC (Mean Corpuscular Hemoglobin Concentration)
MCHC (Mean Corpuscular Hemoglobin Concentration)
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Hematocrit
Hematocrit
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RDW (Red Cell Distribution Width)
RDW (Red Cell Distribution Width)
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Causes of Anemia
Causes of Anemia
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Chronic pancytopenia
Chronic pancytopenia
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Hypocellular bone marrow
Hypocellular bone marrow
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Normochromic, normocytic, hyporegenerative anemia
Normochromic, normocytic, hyporegenerative anemia
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Low reticulocyte count
Low reticulocyte count
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Neutropenia, not lymphopenia
Neutropenia, not lymphopenia
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Aberrant autoreactive T lymphocytes
Aberrant autoreactive T lymphocytes
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Bone marrow failure
Bone marrow failure
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Trilinear cytopenia
Trilinear cytopenia
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Monolinear cytopenia
Monolinear cytopenia
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Pure red blood cell aplasia
Pure red blood cell aplasia
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Myelodysplastic syndromes
Myelodysplastic syndromes
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Gene therapy
Gene therapy
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Ex-vivo gene therapy
Ex-vivo gene therapy
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Allogenic hematopoietic stem cell transplantation
Allogenic hematopoietic stem cell transplantation
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Paroxysmal nocturnal hemoglobinuria (PNH)
Paroxysmal nocturnal hemoglobinuria (PNH)
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CAR-T cells
CAR-T cells
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Allotransplantation
Allotransplantation
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Graft-versus-host disease (GvHD)
Graft-versus-host disease (GvHD)
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Graft-versus-leukemia effect
Graft-versus-leukemia effect
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Suicide gene therapy
Suicide gene therapy
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Chimeric Antigen Receptor (CAR)
Chimeric Antigen Receptor (CAR)
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Natural T cell receptor (TCR)
Natural T cell receptor (TCR)
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CAR-T cell target requirement
CAR-T cell target requirement
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Antigen loss
Antigen loss
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Cytokine release syndrome (CRS)
Cytokine release syndrome (CRS)
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Neurotoxicity
Neurotoxicity
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Tocilizumab
Tocilizumab
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CAR-T cell persistence
CAR-T cell persistence
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CAR-T cell multi-specificity
CAR-T cell multi-specificity
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Myeloproliferative Neoplasms (MPN)
Myeloproliferative Neoplasms (MPN)
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WHO MPN Classification
WHO MPN Classification
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Philadelphia Chromosome
Philadelphia Chromosome
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Bcr-Abl Tyrosine Kinase
Bcr-Abl Tyrosine Kinase
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Imatinib (Gleevec)
Imatinib (Gleevec)
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BCR-ABL transcript monitoring
BCR-ABL transcript monitoring
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Chronic Phase of CML
Chronic Phase of CML
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CML Diagnostic Workup: CBC
CML Diagnostic Workup: CBC
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FISH (Fluorescent In Situ Hybridization)
FISH (Fluorescent In Situ Hybridization)
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Conventional Cytogenetics
Conventional Cytogenetics
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Thrombocytosis
Thrombocytosis
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Second Level MPN Diagnostic
Second Level MPN Diagnostic
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Thrombohemorrhagic
Thrombohemorrhagic
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Budd-Chiari Syndrome
Budd-Chiari Syndrome
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General Thrombotic Risk Factors
General Thrombotic Risk Factors
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Erythropoietin (EPO) Test
Erythropoietin (EPO) Test
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Polycythemia Vera (PV) Diagnostic Criteria
Polycythemia Vera (PV) Diagnostic Criteria
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Essential Thrombocythemia (ET) Diagnostic Criteria
Essential Thrombocythemia (ET) Diagnostic Criteria
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Primary Myelofibrosis (PMF) Diagnostic Criteria
Primary Myelofibrosis (PMF) Diagnostic Criteria
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Gene Addition
Gene Addition
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Gene Subtraction
Gene Subtraction
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Gene Correction
Gene Correction
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Gene Therapy (Definition)
Gene Therapy (Definition)
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Gene Transfer
Gene Transfer
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Vectors
Vectors
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Integrating Vectors
Integrating Vectors
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Non-integrating Vectors
Non-integrating Vectors
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Genome Editing Tools
Genome Editing Tools
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Non-Homologous End Joining (NHEJ)
Non-Homologous End Joining (NHEJ)
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Homologous Directed Repair (HDR)
Homologous Directed Repair (HDR)
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Base Editing
Base Editing
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ADA-SCID
ADA-SCID
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ERT with PEG-ADA
ERT with PEG-ADA
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Retroviral Vector Integration
Retroviral Vector Integration
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Lentiviral Vector Integration
Lentiviral Vector Integration
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Lysosomal Storage Disease
Lysosomal Storage Disease
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Study Notes
Gene Therapy Basics
- Gene therapy is the manipulation of genes to treat, cure, or prevent human disorders, mainly focusing on hematopoietic stem cell-based approaches.
- It involves using nucleic acids (DNA or RNA) to achieve a therapeutic effect.
- Ex vivo gene therapy involves harvesting target cells from a patient, genetically modifying them in the lab, and re-infusing them to achieve a clinical effect through protein expression or lack thereof.
Gene Therapy Strategies
- Gene addition involves adding a therapeutic gene to substitute for a defective or missing gene.
- Gene addition can introduce a new gene, like CAR, to T lymphocytes.
- Gene subtraction inhibits the expression or translation of a protein, using RNAi or antisense RNAs.
- More efficient gene subtraction disrupts coding or regulatory DNA sequences via genome editing or modifies a gene's epigenetic context for silencing.
- Gene correction modifies a mutated gene to transform it into the wild type, combining gene addition and subtraction in one manipulation.
- Genome editing approaches such as homologous recombination, CRISPR editing, base editing, and prime editing facilitates this.
- RNA approaches can also be used for gene correction to induce exon skipping.
- Gene therapy involves using gene-modified cells or vectors in vivo for therapeutic purposes
- Gene transfer refers to transferring, eliminating, or correcting genetic material in a cell to gain a missing function.
- Gene transfer is largely used in research, while gene therapy is meant to be used to find therapies for diseases in a patient.
Gene Therapy Milestones and Vectors
- Discovering and isolating disease-causing genes are crucial for effective gene therapy
- Vectors are essential tools for inserting genes of interest into cells
- Integrating vectors are used for stem cells to ensure long-term gene maintenance and expression in their progeny
- Gamma retroviral vectors were first used, but lentiviral vectors are now preferred for integration due to high efficiency and integration profile
- Lentiviral vectors can infect human cells and hematopoietic cells like CD4 T lymphocytes, monocytes, and macrophages such as HIV-1
- Other vectors exist that are highly efficient transductors, but less able to integrate to HIV-1
- Non-integrating vectors, such as adenoviral vectors or herpes virus, are currently used mainly in in-vivo gene therapy approaches
- Non-viral approaches, like using transposons, are less efficient but much less expensive
Genome Editing Technology
- Gene editing can be achieved using ZFNs, TALEN, and CRISPR, using artificial molecules composed of two moieties.
- One moiety is designed to target a specific genomic DNA area.
- The other moiety, a nuclease, induces a DNA double-strand break.
- CRISPR-Cas9 uses RNA and a Cas9 protein.
- The process involves a guide RNA complementary to the target genome region and a Cas9 protein inducing a DNA double-strand break
- A Nobel prize was awarded for the development of CRISPR
- Once double strand break, there are 3 outcomes
- The cell cannot repair which means it would die
- The second possibility, non-homologous end joining
- The cell could copy an exact base using the other chromosome
- The other chromosome makes an exact base that allows it to repair DNA by adding donor DNA template
DNA Repair Mechanisms
- Following a DNA double-strand break, cells may undergo non-homologous end joining (NHEJ) a "quick and dirty" repair method that can disrupt gene function through insertions or deletions.
- Homologous directed repair (HDR) is more precise but less efficient, using a donor DNA template with homologous flanking regions for gene correction
- Genome editing technology allows for gene disruption via NHEJ or gene correction via HDR, moving gene therapy towards gene substitution and biological function alteration.
- Base editing involves modifying multiple genes at the same time with high precision
- Inducing simultaneous DNA double-strand breaks in different chromosomes poses translocation risks.
- Base editing achieves gene disruption without double-strand breaks, using Cytosine Base Editors (CBE) or Adenine Base Editors (ABE) to change single bases
- While base editing is cleaner, it cannot currently perform gene correction
Ex-vivo Gene Therapy Procedure
- Ex vivo gene therapy typically involves harvesting hematopoietic stem cells due to their accessibility.
- HSCs are harvested and then isolated, cultured, and exposed to genome editing tools before being re-infused into the patient.
- HSCs will distribute themselves in bone marrow niches
- The procedure could involve leukapheresis and IV infusions
- This can treat numerous diseases except those in which there is the need to target cells not in the hematopoietic system
- Vectors can be designed to target certain tissue which started with the liver.
Clinical Achievements in Gene Therapy
- Retroviral vectors can be used to treat primary immunodeficiencies like ADA-SCID, WAS, X-SCID and chronic granulomatosis disease
- Lentiviral vectors can also be used to treat Beta Thalassemia and Sickle Cell Disease and correct phenotype
- Retroviral and Lentiviral vectors can be used to treat adoptive cancer immunotherapy, ALL, B lymphoma, MM
- Editing ZFNs, TALEN and CRISPR can be used in HIV infection and CCR5 disruption
- AAV has been used in the eye like Leber’s Congenital Amaurisis
- AAV, Cas 9 and liver are used in Transthyretin Amyloidosis
- Oncolytic viruses are use as gene therapy for cancer.
HSC and Gene Therapy History
- 1957 saw the first allogenic transplants
- 1992 saw the hematopoietic stem cell based gene therapy for ADA-SCID, using retroviral vectors.
Severe Combined Immunodeficiency (SCID)
- Severe combined immunodeficiency (SCID) is a primary immunodeficiency involving alterations in adaptive and/or innate immunity development and function
- SCID leads to heightened infection susceptibility, autoimmunity risk, and increased cancer risk
- ADA-SCID is an exemple as its defect impacts both B and NK cells
- Gene alteration can be on T cells, Perforin or CD40
- Then Hematopoietic stem cell based gene therapy is used
- HSC gene therapy is used because patients have active infections from allogenic transplantation with HSC
HSC Gene Therapy Overview
- HSC gene therapy follows strict rules as part of the Advanced Therapy Medicinal Product (ATMP) group, which includes cell and tissue engineering
- The Process inolves mobilization/harvest of bone marrow followed by CD34 selection.
- Afterwards perform transduction and then infuse the cells fresh, test using quality controll to ensure quality of the product.
ADA-SCID
- ADA-SCID causes a defect that accumulates toxic metabolites that impair immunity and lead to organ alterations.
- ERT with PEG-ADA requires weekly injections and may provide inadequate immune reconstitution
- Using Stem cells with wt adenosine are a big factor to help
ADA Treatment.
- Long-Term Enzyme replacement helps
- Allogeneic transplant could be alternative
- Genetic therapy is also help
- ADA genetic therapy began 1995
- Systemized in that patient has high number of blood cells.
- Since 2000 the over all survivle is 100% for those treated patients.
HSC - Clinical
- Gamma retroviral were abandon
- For other diseases,retro transformation caused stem cell to tranform
- Translocation usually happens, this means random for every trans cell.
- Vector have own integration, where the regulatory and promoters meet to make more genes and proteins
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