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Questions and Answers
What does the term 'titer' refer to in virology?
What does the term 'titer' refer to in virology?
Monogenic disorders are caused by multiple genes.
Monogenic disorders are caused by multiple genes.
False
What is a 'carrier vector' in the context of gene therapy?
What is a 'carrier vector' in the context of gene therapy?
A vehicle used to deliver therapeutic genes to target cells.
Gene therapy often utilizes a _____ that has been genetically altered to carry normal human DNA.
Gene therapy often utilizes a _____ that has been genetically altered to carry normal human DNA.
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Match the following terms with their definitions:
Match the following terms with their definitions:
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What is a key advantage of using autologous cells in ex vivo therapy?
What is a key advantage of using autologous cells in ex vivo therapy?
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Allogeneic cells carry a high risk for graft-vs-host disease.
Allogeneic cells carry a high risk for graft-vs-host disease.
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Name a challenge associated with autologous cell manufacturing.
Name a challenge associated with autologous cell manufacturing.
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Patisiran is a liposomal siRNA specifically targeting __________.
Patisiran is a liposomal siRNA specifically targeting __________.
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What does ALPPL2 specifically target in cancer therapy?
What does ALPPL2 specifically target in cancer therapy?
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SynNotch CAR-T cells have shown lesser efficacy compared to traditional CAR-T cells in vivo.
SynNotch CAR-T cells have shown lesser efficacy compared to traditional CAR-T cells in vivo.
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What is the ClinicalTrials.gov Identifier for the current status of the ALPPL2 study?
What is the ClinicalTrials.gov Identifier for the current status of the ALPPL2 study?
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The _____ of technological innovation in gene and cell therapy is outpacing the ability to safely move candidates forward.
The _____ of technological innovation in gene and cell therapy is outpacing the ability to safely move candidates forward.
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Match the following therapies with their descriptions:
Match the following therapies with their descriptions:
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What type of DNA molecule is a plasmid?
What type of DNA molecule is a plasmid?
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Bacterial transformation is a process by which bacteria can take up foreign genetic material from the environment.
Bacterial transformation is a process by which bacteria can take up foreign genetic material from the environment.
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Who won the Nobel Prize in 1958 for their contributions to microbiology?
Who won the Nobel Prize in 1958 for their contributions to microbiology?
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Plasmids can replicate independently of __________ DNA in the cell.
Plasmids can replicate independently of __________ DNA in the cell.
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What is a limitation of plasmid-mediated gene delivery?
What is a limitation of plasmid-mediated gene delivery?
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Viral vectors are always integrating viruses.
Viral vectors are always integrating viruses.
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Adenoviruses primarily cause __________ infections in humans.
Adenoviruses primarily cause __________ infections in humans.
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What is a disadvantage of using lentiviral vectors compared to gammaretroviral vectors?
What is a disadvantage of using lentiviral vectors compared to gammaretroviral vectors?
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Self-inactivating (SIN) vectors can help reduce the risk of genotoxicity.
Self-inactivating (SIN) vectors can help reduce the risk of genotoxicity.
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What severe condition did Jesse Gelsinger suffer from that led to his death after gene therapy?
What severe condition did Jesse Gelsinger suffer from that led to his death after gene therapy?
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Lentiviral vectors may integrate less frequently upstream of __________.
Lentiviral vectors may integrate less frequently upstream of __________.
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Match the following gene therapy cases with their outcomes:
Match the following gene therapy cases with their outcomes:
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What is a major feature of adeno-associated vectors (AAV)?
What is a major feature of adeno-associated vectors (AAV)?
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Adeno-associated vectors are known to integrate into the genome of host cells at high frequencies.
Adeno-associated vectors are known to integrate into the genome of host cells at high frequencies.
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Which of the following statements about CRISPR/Cas9 is true?
Which of the following statements about CRISPR/Cas9 is true?
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What disease is directly associated with a deficiency in clotting factor IX?
What disease is directly associated with a deficiency in clotting factor IX?
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Lentiviral vectors are completely free from the risk of mutagenesis.
Lentiviral vectors are completely free from the risk of mutagenesis.
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What is a common safety concern associated with gene therapy?
What is a common safety concern associated with gene therapy?
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AAV can transduce cells as double stranded circular ______.
AAV can transduce cells as double stranded circular ______.
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Match the clinical examples to their related conditions:
Match the clinical examples to their related conditions:
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What percentage of patients may be excluded from AAV treatment due to pre-existing immunity?
What percentage of patients may be excluded from AAV treatment due to pre-existing immunity?
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The use of Adeno-associated vectors results in high cytotoxicity.
The use of Adeno-associated vectors results in high cytotoxicity.
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Name one structural component that mediates viral entry to cells.
Name one structural component that mediates viral entry to cells.
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Study Notes
Gene Therapies Overview
- Gene therapy aims to treat diseases by inserting, altering, or removing genes within cells and tissues. Controversies exist about the exact definition, as its scope evolves.
- Methods include gene suppression using RNAi or antisense oligonucleotides, increasing/restoring gene expression, and gene modification via genome editing.
Lecture Objectives
- MAIN: Understand the general concept and scope of gene therapy.
- Learn the different methods of gene delivery/correction.
- Identify the advantages, disadvantages, and limitations of each delivery system.
- SUPPLEMENTARY: Delve into the clinical potential of gene therapy.
- Explore the ethical considerations and challenges.
- Gain enthusiasm for the topic.
Lecture Content
- PART I: General introduction and categorization of gene therapy.
- PART II: Detailed explanation of gene delivery/correction systems.
- PART III: Example of proof-of-principle to clinical trials.
Definition of Gene Therapy
- Gene therapy involves inserting, altering, or removing genes within an individual's cells and tissues to treat diseases.
- Various approaches are considered, but the fundamental idea is consistent.
Classifying Gene Therapy
- Type of Disease: Genetic or acquired.
- Delivery Vehicle: Integrating or non-integrating vectors.
- Type of Administration: In vivo or ex vivo.
Origin of Gene Therapy
- Proposed in 1972, the initial concept involved using exogenous DNA to replace defective DNA causing genetic defects.
- The first clinical trial in 1990, involved a four-year-old girl with an immune deficiency at the NIH.
Terminology
- Transformation: Transfer of naked DNA into bacteria.
- Transfection: Transfer of naked DNA into cells.
- Infection: Infecting with a wild-type virus.
- Transduction: Foreign DNA is introduced into another cell via a viral vector.
- Titer: A way to express the concentration of viral particles (Infectious Units per ml—IU/ml).
Genetic Background of Diseases
- Genetic Factors: Monogenic (caused by one gene) and polygenic (requires multiple genes).
- Environmental Factors: Infectious diseases, and factors contributing to complex conditions like cancers, chronic diseases.
Types of Faulty Genetics
- Correct Function: The gene and protein function normally.
- Loss-of-Function: The gene or resulting protein is ineffective.
- Gain-of-Function: The gene or protein causes unwanted changes.
Basis of Gene Therapy
- A carrier vector, often a modified virus, delivers the therapeutic gene to target cells.
- The vector inserts the functional gene into the cell to achieve a normal state.
Gene Delivery
- Viruses naturally deliver their genetic material, providing a template for therapeutic gene delivery.
- Researchers can modify viruses to carry therapeutic genes safely.
Ex Vivo or In Vivo
- Ex Vivo: The technique involves taking cells out of the body, modifying them, and returning them to the body.
- In Vivo: The technique involves directly introducing the gene therapy directly into the patient's body.
Ex Vivo: Self or Non-self
- Autologous: Using the patient's own cells; no rejection issues but more costly and time-consuming.
- Allogeneic: Using cells from a donor; potential for rejection and graft-vs-host disease.
Ex Vivo: Advantages and Challenges
- Autologous: Patient-specific, no rejection/graft-vs-host, repeatable doses.
- Autologous Challenges: High manufacturing/quality testing costs, potential starting material variability.
- Allogeneic Advantages: Larger-scale manufacturing, potentially more reproducible, faster availability (banking).
- Allogeneic Challenges: Potential for graft-vs-host disease, risk for rejection, high cost minimizing steps
Delivery Vehicles: Non-viral
- Liposomes: Lipid-based nanoparticles, enclosing plasmid DNA.
- Solid-Lipid Nanoparticles: The structure encapsulates therapeutic agents.
- Niosomes: Contains non-ionic surfactants, and a cationic lipid, carrying plasmid DNA.
- Polymer nanoparticles: polymer composites enclosing therapeutic payloads.
Non-viral Example (Hereditary Variant Transthyretin Amyloidosis - ATTRv)
- ATTRv is a rare inherited disease causing mutant transthyretin protein misfolding.
- Therapies like Patisiran (a liposomal siRNA) target TTR accumulation.
Non-viral Example (COVID-19 mRNA vaccine)
- mRNA vaccines encode for the virus protein.
- They are injected intramuscularly using lipid nanoparticles.
- This triggers an immunological response.
Plasmids: Excellent gene carriers
- Circular, double-stranded DNA molecules, self-replicating within bacteria.
- Easily modified for human use.
Plasmid production
- Insertion of gene of interest into a plasmid vector.
- This vector allows for the amplification of the desired plasmid.
Plasmid mediated gene delivery
- Low efficiency, only transient expression, difficult with primary cells.
Delivery Vehicles: Viral
- Adenovirus: Non-integrating, high in-vitro/in-vivo transduction and expression, produced in high amounts.
- AAV: Non-integrating, smaller size and lower immunogenicity compared to adenovirus.
- Retrovirus: Integrating.
- Lentivirus: Integrating, unique ability to infect non-dividing cells.
Viral Vectors: Non-Integrating
- Adenovirus: Often causing the common cold. No integration into the host cell genome.
- AAV: Not harmful for humans. DNA does not integrate, but at low frequencies.
Viral Vectors: Integrating
- Retrovirus: Creating double-stranded DNA from RNA. Integrating into host cell chromosomes. Typically only infects dividing cells.
- Lentivirus: A sub-type of retrovirus that is able to infect non-dividing cells
Adenoviral vs. AAV
- Adenoviral vectors are more effective for gene delivery but can cause immune responses in the body.
- AAV vectors have lower immunogenicity and can be used in vivo.
Adenoviral Vectors: Delivery
- Viral attachment and entry. Endocytosis, viral escape. Nuclear import and expression.
Adenoviral Vectors: Major features
- High efficiency of transduction, high levels of expression easily produced in high amounts, infect both dividing and non-dividing cells, rarely integrates into the host cell genome.
Adenoviral Vectors: Major negative features
- Ad5 infection requires CAR expression for binding. Immune response in the patient to previous exposure is a concern.
- Persistent gene expression is difficult in dividing and non-dividing cells due to different mechanisms.
Adeno-associated vectors (AAV)
- Non-integrating, small size, lower immunogenicity.
- Effective delivery of genes in both dividing and non-dividing cells,
- Not pathogenic to humans. Rare integration but possible.
AAV: Major features
- Smaller in size, smaller packing capability, can infect dividing and non-dividing cells, very low immunogenicity and cytotoxicity
AAV's way to the Clinic
Early isolation and cloning, production of recombinant AAV, safety assessments, characterization of structure, and engineering of new capsids.
- Challenges in some individuals experiencing pre-existing immunity.
Clinical examples AAV
- Hemophilia B, retinal disease, Parkinson's, cystic fibrosis, and lysosomal storage diseases.
Clinical Examples: Hemophilia B
- Genetic condition resulting from a deficient clotting factor IX.
- Bioengineered AAV vectors (delivering a functional gene) achieve gain-of-function for factor IX expression, which addresses clotting deficiencies.
Viral Vectors: Integrating
- Retroviruses tend to integrate into host cell genomes, meaning the therapeutic gene is permanently a part of the cell.
- They require active cell division.
- Potential risk of insertional mutagenesis due to genome integration.
Insertional Mutagenesis
- Various viruses and vectors lead to undesirable changes or activation of surrounding genes/elements.
Historical overview of HSC gene therapy
- Early stages of HSC gene therapy research focusing on isolating, cloning, and modifying human stem cells.
- Various approaches tested and refined (various vectors).
- Advances in manufacturing and purification processes improve safety.
Clinical Examples: Retrovirus
- Focus on Primary Immunodeficiencies (PIDs) including different types of SCID (Severe Combined Immunodeficiencies), ADA SCID, Wiskott-Aldrich Syndrome, and Chronic Granulomatous disease.
Clinical example: ADA-SCID
- Rare disorder arising from a defect in ADA gene, affecting lymphocyte production.
- Severe infections, immune problems, early death without treatment.
ADA-SCID: Gene therapy approach
- Treatment options include matched-related or unrelated bone marrow transplants and PEG-ADA enzyme replacement therapy.
- Approaches to introduce functional ADA into cells are via retroviral vectors
Lentiviral vectors: Major features
- Integration, permanent expression.
- Successful delivery in non-dividing cells due to nuclear transport mechanisms.
- Possible risk of insertional mutagenesis (but lower than gammaretroviral vectors.)
Safety: Lentiviral Vectors
- Tendency to integrate near active genes but less frequently up-stream.
- Less chance of activating adjacent oncogenes compared to others.
Update: Self-Inactivating (SIN) vectors
- Removal of endogenous strong enhancer elements helps reduce genotoxicity.
- Applies to lentiviral and gammaretroviral vectors.
Safety concerns (real examples)
- Gelsinger death, Severe problems in X-SCID treatments, and challenges in XLMTM cases.
Gene editing tools
- Using CRISPR technology (which has a high feasibility rating).
- Techniques to create double strand DNA breaks to have effective replacement or inactivation of sequences of interest, often with DNA templates.
CRISPR/Cas9 system
- Utilizing the CRISPR-Cas9 system for genome editing.
- Cas9 protein with gRNA to find and target specific DNA segments, resulting in DNA cleavage.
- The bacterial immune system adapted for gene editing.
CRISPR/Cas9 origins
- Cellular immune system.
- Finding and disrupting target DNA.
- This is a directed gene editing process.
CRISPR/Cas9 way to the clinic
- Discovery/identification of CRISPR, fundamental relationships, applications.
- First human trials including HIV treatment and blindness therapies.
- Recent advancements with germline editing.
CRISPR/Cas9 versions
- Gene editing, gene regulation/epigenome editing, chromatin imaging.
- CRISPR tools/systems developed for various purposes.
CRISPR/Cas9 administration
- In vivo (directly into the body) or ex vivo (taking cells out modifying them then putting them back).
- Using viral or non-viral vectors for delivery.
CRISPR/Cas9 delivery methods (ex vivo)
- Plasmid with Cas9 and sgRNA, Cas9 mRNA + sgRNA, Cas9 Protein with sgRNA complex- various delivery methods exist to optimize treatment for each patient and disease.
CRISPR/Cas9 example (Sickle cell disease – SCD, Beta-thalassemia)
- Inherited blood disorders.
- Gene editing approaches like CRISPR/Cas9 to correct faulty hemoglobin genes.
- Addressing disease severity, and potential long-tail implications.
CRISPR/Cas9 example processes
- Standardized processes for bone marrow transplants, isolating and processing CD34+ cells (via electroporation).
CRISPR/Cas9: What's cooking?
- Clinical trials for various diseases including hemoglobinopathies, chronic bacterial infections, protein folding disorders and more.
Clinical Examples: Acquired disease (Cancer)
- Cancer treatment using tumor-specific T cell receptors (TCR).
Immune cells and Cancer
- Cellular mechanisms and systems involved with the immune system's response to cancerous cells including naîve T cells, activated T cells, Dendritic cells, B cells, and NK cells.
The Chimeric Antigen Receptor (CAR)
- Antibody portion binds to tumor antigens and a T-cell portion delivers the signal in the CAR T-cell.
CARs differ from the T cell receptor
- CARs recognize tumor cells independent of MHC.
- CARs can target non-protein surface molecules.
- CARs only recognize antigens expressed on the cell surface, unlike TCRs, which also recognize those presented by MHC molecules
What is CAR-T cell therapy?
- Genetically modified T cells to target and destroy cancerous cells.
- T-cells are engineered to exhibit anti-tumor function.
How are CAR-T cells produced?
- Acquire T-cells from the patient. Genetically engineer them with CAR proteins.
- Expand that modified cell population.
- Administer CAR-T cells to the patient.
- Targeted cancer cell destruction.
CAR-T cell therapy in B cell Malignancies
- Targeting B-cell malignancies using CAR-T cells (like Leukemia and Lymphoma), identifying affected cells
Patient "Proof-of-Principle"
- Reported successes in acute lymphoblastic leukemia.
- Demonstrates success in clinical trials.
Clinical Success of CAR T cell therapy
- KYMRIAH® (Tisagenlecleucel) approved for pediatric and adolescent acute lymphoblastic leukemia (ALL).
- Complete remissions in cancer cases/patients with prolonged remissions.
CAR-T cell therapy: Major features
- Pros: Personalized treatment, lasting immunity, no graft-versus-host disease risk.
- Cons: High cost, time-consuming production method, risk of adverse events (like cytokine release syndrome).
Broadening CAR-T cell Therapy
- Growth in trials across multiple cancer types (across the decades).
- Geographical spread of trials (particularly in the United States, China, and the United Kingdom).
Functional Challenges in CAR T cell therapy
Factors like trafficking and microenvironment interaction influence T-cell function and clinical outcome during treatment.
- Other factors include control, recognition, and proliferation/persistence capabilities.
Next-generation CAR-T cells
- Research developing further refinement of CAR-T cell technology. Trials progressing.
Multi-antigen recognition of tumors
- Multiple targets recognized by CARs to enhance anti-tumor efficacy.
- Multiple targets help inform understanding of CAR therapy, safety and efficacy.
New class of synthetic environmental sensor
- New approach to synthetic sensors for improving CAR T-cell response and efficacy to tumor targets.
Developing new clinically relevant circuit CAR-T cells
- SynNotch CAR circuits for enhancing solid tumor recognition.
- Research in mouse models.
Mesothelioma
- Heavily associated with asbestos exposure.
- Extremely aggressive with limited treatment options.
- Three subtypes (epithelioid, sarcomatoid, biphasic).
ALPPL2: A New Tumor-Specific Antigen
- A newly identified tumor antigen.
- High expression observed in various cancers (mesothelioma, seminoma, gastric, etc.) but not in normal tissues
Combinatorial targeting using ALPPL2
- Combination of different antigen targeting strategies.
- Specific response to cancer cells.
SynNotch CAR-T cells superior efficacy in vivo
- Demonstrated superior efficacy in animal models (M28 and M2L-KO).
ALPPL2 synNotch for other tumor types
- Evaluated on other cancers.
- High efficacy observed in some cases (SK-OV-3, K562)
Next step toward the clinic
- Further design of synthetic receptors for programmed gene regulation.
Problems with SynNotch
- Difficulty with specific antigen binding, treatment, delivery, and regulation.
- Humanization is underway to address these problems.
New humanized version
- Humanized versions of ALPPL2 SNIPR to target SKOV-3 cancer cells for an improved response.
Current status
- Arsenalbio's Phase I clinical trial of AB-1015 in treating ovarian cancer recently begun.
Concluding Remarks
- Technological advancements in gene and cell therapy outpace regulatory frameworks.
- Personalized therapies and future innovation are essential.
Three essential tools for human gene therapy
- Adeno-associated viral (AAV)
- Lentiviral vector
- Gene editing components important for treatment.
Suggested Reading
- Comprehensive list of relevant scientific articles.
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Description
Test your knowledge on key concepts in gene and cell therapy. This quiz covers various terms, definitions, and therapeutic approaches, including the use of autologous cells and the role of carrier vectors in gene therapy. Dive into the details of challenges and innovations within this rapidly evolving field.
