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Questions and Answers
What does the term 'Titer' refer to?
What does the term 'Titer' refer to?
Monogenic disorders are caused by multiple genes.
Monogenic disorders are caused by multiple genes.
False (B)
What is the purpose of a 'carrier vector' in gene therapy?
What is the purpose of a 'carrier vector' in gene therapy?
To deliver the therapeutic gene to the patient's target cells.
The combination of environmental and genetic factors is often associated with _______.
The combination of environmental and genetic factors is often associated with _______.
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Match the following terms with their definitions:
Match the following terms with their definitions:
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Which of the following statements is true regarding autologous cells?
Which of the following statements is true regarding autologous cells?
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Allogeneic cells pose a risk of graft-vs-host disease.
Allogeneic cells pose a risk of graft-vs-host disease.
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What genetic condition is addressed by the treatment Patisiran?
What genetic condition is addressed by the treatment Patisiran?
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Patisiran is a liposomal siRNA that targets ________ to treat ATTRv.
Patisiran is a liposomal siRNA that targets ________ to treat ATTRv.
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Match the following types of cell sources with their respective advantages:
Match the following types of cell sources with their respective advantages:
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What is the most significant challenge in gene and cell therapy as mentioned?
What is the most significant challenge in gene and cell therapy as mentioned?
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The SynNotch CAR-T cells show inferior efficacy compared to other treatment methods.
The SynNotch CAR-T cells show inferior efficacy compared to other treatment methods.
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What is the ClinicalTrials.gov Identifier for the new humanized version of ALPPL2?
What is the ClinicalTrials.gov Identifier for the new humanized version of ALPPL2?
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One essential tool for human gene therapy is __________.
One essential tool for human gene therapy is __________.
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Match the following topics with their corresponding descriptions:
Match the following topics with their corresponding descriptions:
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What is the primary goal of Gene Therapy?
What is the primary goal of Gene Therapy?
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The first gene therapy clinical study was conducted in 2000.
The first gene therapy clinical study was conducted in 2000.
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What disease was targeted in the first gene therapy clinical study?
What disease was targeted in the first gene therapy clinical study?
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Gene Therapy can involve the use of RNAi for gene ________.
Gene Therapy can involve the use of RNAi for gene ________.
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Match the terms related to Gene Therapy with their definitions:
Match the terms related to Gene Therapy with their definitions:
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Which of the following represents a limitation of Gene Therapy?
Which of the following represents a limitation of Gene Therapy?
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Gene Therapy is a defined and settled field without any controversies.
Gene Therapy is a defined and settled field without any controversies.
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Who was the patient in the first gene therapy clinical study?
Who was the patient in the first gene therapy clinical study?
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What is the primary purpose of CRISPR/Cas9 in medicine?
What is the primary purpose of CRISPR/Cas9 in medicine?
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CRISPR/Cas9 technology can only be used to treat infectious diseases.
CRISPR/Cas9 technology can only be used to treat infectious diseases.
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What does CAR-T cell therapy involve?
What does CAR-T cell therapy involve?
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Sickle cell disease affects millions globally due to errors in the genes for _______.
Sickle cell disease affects millions globally due to errors in the genes for _______.
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Match the following diseases with their causes:
Match the following diseases with their causes:
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Which of the following is a method used in CRISPR/Cas9?
Which of the following is a method used in CRISPR/Cas9?
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The introduction of tumor-specific T cell receptors is a clinical application of CRISPR/Cas9.
The introduction of tumor-specific T cell receptors is a clinical application of CRISPR/Cas9.
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What is a plasmid?
What is a plasmid?
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What role do artificial receptors play in CAR-T cell therapy?
What role do artificial receptors play in CAR-T cell therapy?
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Bacterial transformation involves the uptake of foreign genetic material from the environment.
Bacterial transformation involves the uptake of foreign genetic material from the environment.
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What is the disadvantage of plasmid-mediated gene delivery?
What is the disadvantage of plasmid-mediated gene delivery?
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Adenovirus is a class of virus with __________ genomes.
Adenovirus is a class of virus with __________ genomes.
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Which of the following statements about plasmids is true?
Which of the following statements about plasmids is true?
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Plasmid-mediated gene delivery results in permanent integration of genetic material into the host genome.
Plasmid-mediated gene delivery results in permanent integration of genetic material into the host genome.
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The uptake of foreign genetic material in bacteria is referred to as __________.
The uptake of foreign genetic material in bacteria is referred to as __________.
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Study Notes
Gene Therapies
- Gene therapy is the insertion, alteration, or removal of genes within an individual's cells to treat disease. There is ongoing debate about what exactly constitutes gene therapy.
- Various methods are used for gene delivery/correction including suppression with RNAi or antisense oligonucleotides, increased/restored expression by introducing a gene, and gene modification(s) with genome editing.
Who Am I?
- Axel Hyrenius Wittsten is a researcher in synthetic immunology at Lund University, Sweden.
- He has a PhD from Lund University
- He has conducted postdoctoral research at the University of California, San Francisco (UCSF), USA
Lecture Objectives
- Main Objectives: Understand the overall concept and scope of gene therapy, learn the fundamental systems used for gene delivery/correction, and identify the advantages, disadvantages, and limitations of each system.
- Supplementary Objectives: Gain insight into the clinical potential of gene therapy, understand the ethical considerations surrounding it, and become excited about its potential.
Lecture Content
- Part I: General introduction and categorization of gene therapies.
- Part II: Detailed explanation of gene delivery systems and their applications.
- Part III: Case study of gene therapy from proof-of-concept to clinical trials.
Definition of Gene Therapy
- Gene therapy modifies an individual's cells or tissues through the insertion, alteration, or removal of genes to cure disease.
- Ongoing debate exists on the scope of what constitutes gene therapy.
Classifying Gene Therapy
- Type of Disease: Can be Genetic or Acquired.
- Delivery Vehicle: Can be Integrating or Non-integrating.
- Type of Administration: Can be In Vivo or Ex Vivo.
Origin of Gene Therapy
- Early proposals (1972) suggested using exogenous DNA to replace defective DNA in genetically affected individuals.
- The first gene therapy clinical study (1990, NIH) involved a 4-year-old girl (Ashanti DeSilva) with an immune deficiency.
Terminology
- Transformation: Transfer of naked DNA into bacteria.
- Transfection: Transfer of naked DNA into cells.
- Infection: Infecting a cell with a wild-type virus.
- Transduction: Foreign DNA is introduced to another cell via viral vector.
- Titer: Method of expressing concentration of viral particles or genetic material within them (Infectious Units per ml, IU/ml).
Genetic Background of Diseases
- Genetic factors: Inherited disorders, including monogenic (caused by one gene) and polygenic disorders (caused by many genes), are responsible for some diseases.
- Environmental factors: Infectious diseases are one category of diseases caused by environmental influences.
- Diseases can also result from a combination of environmental factors and genetic factors. For instance, some cancers and chronic diseases have these complex origins.
Types of Faulty Genetics
- Loss-of-function: Genetic defects that result in a gene or protein not functioning as intended. The specific protein no longer performs its normal function.
- Gain-of-function: Genetic defects that result in a gene or protein exhibiting unintended or excess activity. The protein does something unexpectedly.
Basis of Gene Therapy
- A carrier vector is employed to deliver a therapeutic gene to target cells.
- Most vectors are viruses that have been genetically altered to carry the desired normal human DNA.
- The vector delivers the gene into target cells to generate functional proteins, restoring cells to a normal state.
Gene Delivery
- Viruses are modified to deliver therapeutic genes to cells.
- The genetic material in the vector is inserted into cells, and the generation of functional proteins restores the cells to a normal state.
Ex Vivo or In Vivo
- In vivo: Viral vectors are directly introduced to the target organ inside the patient.
- Ex vivo: Cells are extracted in a lab setting, genetically modified, and then returned to the patient.
Ex Vivo: Self or Non-self
- Autologous: Patient's own cells are used. No rejection risk. Higher costs.
- Allogeneic: Cells from a donor are used. Risk of graft-vs-host disease. May require fewer manufacturing steps, potentially lower costs.
Delivery Vehicle: Non-viral
- Liposome-based nanoparticles: Cationic lipids and neutral phospholipids form spheres to deliver therapeutic material.
- Solid-lipid nanoparticles: Form stable cores to enclose therapeutic materials.
- Niosomes: Surfactants arrange around an aqueous core to encapsulate treatment material.
- Polymer-based nanoparticles: Polymeric vectors can carry therapeutic material and DNA.
### Non-viral example (Hereditary variant transthyretin amyloidosis)
- Hereditary variant transthyretin amyloidosis (ATTRv) involves protein misfolding to form aggregates and amyloid deposition in the body.
- Patisiran is a liposomal siRNA that targets TTR, decreasing TTR accumulation, and potentially improving clinical symptoms.
Non-viral example (COVID-19 mRNA vaccines)
- COVID-19 mRNA vaccines use modified mRNA to induce immune responses to SARS-CoV-2 spike protein.
- Nucleoside-modified mRNA and circular mRNA, as well as lipid nanoparticle-mRNA are different types of mRNA used.
Plasmids: Excellent gene carriers
- Plasmids are circular double-stranded DNA molecules that replicate independently in bacteria.
- Plasmids can carry foreign DNA, making them useful gene carriers.
- Naked DNA is useful in gene delivery.
Plasmid mediated gene delivery
- Low transfer efficiency means that plasmids may deliver genes inefficiently.
- They provide only transient expression.
- They are sometimes difficult to use on primary cells.
Delivery Vehicle: Viral
- Size, genome, packaging capacity, transduction efficiency, integration, and expression differ among various viral vectors.
- Adenovirus, adeno-associated virus, retrovirus, and lentivirus are different types of viral vectors.
- Safety levels and immunogenicity vary for the different viral vectors.
Viral vectors: Non-integrating viruses
- Adenovirus: Double-stranded DNA viruses with a common cold genome. Their DNA does not integrate into the host cell's genome.
- Adeno-associated virus (AAV): Small, single-stranded DNA virus that are often replication-defective.
Viral vectors: Integrating viruses
- Retrovirus: Can create double-stranded DNA copies from its RNA genome which can integrate into host cells' chromosomes. It can only infect dividing cells.
- Lentivirus: A sub-type of retrovirus that has the unique ability to infect both dividing and non-dividing cells.
Adenoviral vectors
- Adenovirus is a double-stranded DNA virus commonly causing respiratory, intestinal, and eye infections.
- The transferred adenoviral gene does not integrate into the host's genome; so gene expression is transient.
- It is easy to produce in sufficient quantities, can infect both dividing and non-dividing cells and generally does not integrate into the host's genome, creating a high level of efficacy.
Adenoviral vectors: Delivery
- Viral entry, endocytosis, escape from endosome, trafficking to nucleus, docking at nuclear pore complex, DNA import/expression are part of the delivery route.
- These steps result in adenoviral vector delivery in cells.
Adenoviral vectors: Major positive features
- High efficiency of transduction.
- High levels of transgene expression.
- Easily produced in high amounts.
- Infect both dividing and quiescent cells.
- Rarely integrates into the host genome.
Adenoviral vectors: Major negative features
- Adenovirus type 5 requires a specific receptor.
- Many individuals have developed an immune response to this type of virus.
- Persistent gene expression is challenging due to a lack of integration.
Adeno-associated vectors (AAV)
- A class of small, single-stranded DNA viruses not usually pathogenic to humans.
- AAV DNA does not integrate into the genome of the host cell but can integrate at low frequencies.
AAV: Major Features
- Smaller than adenoviruses.
- Transduce both dividing and non-dividing cells.
- Low immunogenicity and cytotoxicity.
- Gene is delivered as double stranded circular episomes that can either integrate or be transient.
AAVs way to the Clinic
- Summarizes the advances and challenges in using AAVs as gene therapy vectors from the isolation to clinical trials.
Clinical examples: AAV
- Details of clinical trials for AAV treatments in different diseases (e.g., hemophilia B, retinal, Parkinson's, cystic fibrosis, and lysosomal storage).
Clinical examples: Hemophilia B
- Hemophilia B is linked to a clotting factor IX deficiency. Bioengineered AAV vectors deliver a fix IX Padua transgene to the liver.
Viral vectors: Integrating virus
- Retrovirus: Retroviral genomes create double-stranded DNA versions of their RNA genomes, which then integrate into the host cell's chromosomes.
- Lentivirus are a subset of retroviruses able to infect non-dividing cells.
Recombinant integrating vectors
- Viral components are separated onto "packaging" plasmids and the target transgene onto a "transfer" plasmid. In this way, genetic material can be separated and incorporated efficiently.
Producing integrating vectors
- The creation of integrating vector constructs from recombinant viral vectors is discussed.
Examples of Integrating vectors
- Mechanisms for creating vectors and their implications for delivering genes.
Retroviral vectors: Major features
- Retroviral vector genome integrates into the host cell genome to elicit a sustained response.
- Host cell's division is a necessity for this type of vector delivery.
- Integration may cause insertional mutagenesis
Insertional mutagenesis
- Vectors can insert into genes that have critical functions, possibly causing harm.
- This may increase the risk of cancers or other health problems
Historical overview of HSC gene therapy
- Summarizes the historical development of gene therapy targeting hematopoietic stem cells.
Clinical examples: Retrovirus
- Details specific gene therapy clinical examples for primary immunodeficiency disorders.
Clinical example: ADA-SCID
- ADA-SCID is a rare disorder where adenosine deaminase (ADA) is deficient and lymphocytes cannot be created or function normally.
Main symptoms of ADA deficiency
- Pneumonia, chronic diarrhea, and skin rashes.
- Developmental delays are common.
ADA-SCID: Gene therapy approach
- Various approaches for treating ADA-SCID, including homologous bone marrow transplants, and weekly enzyme replacement therapy.
Lentiviral vectors: Major features
- Lentiviral vectors integrate into the host cell genome resulting in persistent transgene expression.
- They can infect both dividing and non-dividing cells.
- Potential risk of insertional mutagenesis exists due to the integration mechanism .
Safety: Lentiviral vectors
- Lentivirus's preferential integration into active genes, which may limit the risk of adverse events compared to random integrations.
- This limits their risk for activating adjacent oncogenes.
Update: Self-Inactivating (SIN) vectors
- Removal of endogenous strong enhancer elements in the vector using a self-inactivating design is discussed as a way to reduce possible genotoxicity.
Safety concerns (real examples)
- Real-world cases highlighting safety concerns (e.g., Gelsinger death, Leukemia in X-SCID patients, and liver failure in other trials).
Gene editing tools
- Various tools for gene editing are discussed, including meganucleases, zinc-finger nucleases, TALENs, and CRISPR/Cas9.
CRISPR/Cas9 system
- The CRISPR/Cas9 gene editing system has revolutionized the field of gene editing, contributing to breakthroughs in medicine and biotechnology.
CRISPR/Cas9 origins
- Bacteria employ a CRISPR/Cas system for defense against viral infection.
- The CRISPR/Cas system has been adapted for targeted genome editing in many different organisms, including humans.
CRISPR/Cas9 way to the clinic
- Shows how CRISPR/Cas9 has advanced to clinical trials for several diseases including HIV-1 , blindness, cancer, and germline editing for human embryos, showing how the science is moving toward clinical application rapidly.
CRISPR/Cas9 versions
- Describes different versions of the CRISPR/Cas9 system, including gene editing, gene regulation, epigenome editing, chromatin imaging.
CRISPR/Cas9 administration
- In vivo and ex vivo strategies for CRISPR/Cas9 delivery are reviewed.
CRISPR/Cas9 delivery methods (ex vivo)
- Various methods for CRISPR/Cas9 delivery in ex vivo settings are reviewed.
CRISPR/Cas9 Example (Sickle cell disease and thalassemia)
- Describes the genetic errors that cause sickle cell disease and thalassemia, and possible treatments using CRISPR/Cas9, which target errors within hemoglobin gene sequences, and their implications.
CRISPR/Cas9 example
- Details example CRISPR/Cas9 processes for existing bone marrow transplants.
CRISPR/Cas9: what's cooking?
- Current clinical trials using CRISPR/Cas9 technology spanning various diseases are shown.
Clinical examples: Acquired disease (Cancer)
- CAR-T cell therapy using tumor-specific T cell receptors to address cancer.
Immune cells and Cancer
- Describes the relationship between the adaptive and innate immune responses against cancer.
The Chimeric Antigen Receptor (CAR)
- Describes the components and construction of CAR.
CARs differ from the T cell receptor
- Highlights the distinct functionality and mechanisms between CARs and T cell receptors.
What is CAR-T cell therapy?
- CAR-T cell therapy involves reengineering a patient's own T cells to recognize and eradicate cancer.
- Genetically altered T cells express artificial receptors that enable them to bind to specific antigens on tumor cells, triggering their destruction.
How are CAR-T cells produced?
- Procedure for producing CAR-T cells from patient samples, cell expansion, and infusion.
CAR-T cell therapy in B cell Malignancies
- Description of CAR-T cell therapy as used for leukemia and lymphoma.
### Patient "Proof-of-Principle"
- Cases where CAR-T cell therapy has been used to address treatment-resistant cancers are described.
Clinical Success of CAR T cell therapy
- Illustrates the use of CAR-T cell therapy as it has progressed and provides results from its approval by the FDA.
CAR-T cell therapy: Major features
- Highlights the advantages and disadvantages of using CAR-T cell therapy.
Broadening CAR-T cell Therapy
- Shows advances in the field, including the increasing number of trials, and comparison between solid and hematologic cancers.
Functional Challenges in CAR T cell therapy
- Summarizes the challenges in CAR-T cell therapy, including trafficking, control, recognition, and proliferation/persistence.
Next generation CAR-T cells: Proof-of-principle to Clinical Trial
- Shows the transition from laboratory development to clinical trials for the next-generation of CAR-T cells.
Multi-antigen recognition of tumors
- Data about the landscape of tumor targets with their variety, and suggested strategies for developing new methods for cancer treatment.
New class of synthetic environmental sensor
- Emphasizes the use of SynNotch in sensing the tumor microenvironment.
Developing new clinically relevant circuit CAR-T cells
- Describes the development of new CAR-T cell circuits.
Mesothelioma
- This cancer is heavily linked to asbestos exposure, has a prolonged incubation period, is aggressive, and has limited options for treatment.
ALPPL2: a new tumor-specific antigen
- Presentation of a new tumor surface marker, ALPPL2.
Combinatorial targeting using ALPPL2
- Methodologies that use ALPPL2 in conjunction with other tumor-specific antigens to induce an immune cascade against tumor and treatment-resistant tumors.
SynNotch CAR-T cells exhibit superior efficacy in vivo
- Discusses the efficacy of SynNotch CAR-T cells in different Mesothelioma subtypes (M28, and M38)
ALPPL2 synNotch for other tumor types
- SynNotch CAR-T cells are also effective for other cancer types.
Next step toward the clinic: Modular design of synthetic receptors for programmed gene regulation in cell therapies
- Future directions for advancing cell and gene therapy technology are summarized.
Problems with SynNotch
- Potential issues associated with the use of SynNotch CARs are highlighted.
New humanized version
- Specific antigen targeting of SKOV-3 ovarian cancer cells using humanized receptor technology.
Current status
- Recent progress on the development of engineered CAR T cell therapies for ovarian cancer.
Concluding remarks
- The speed of technological innovation in gene therapies is much faster than our ability to ensure they are safe and effective. This rapid change requires the development of regulatory controls to safely introduce these new therapies.
Three essential tools for human gene therapy
- Identification of critical tools in gene therapy and their applications.
Suggested Reading
- Listing of key resources for further reading and study.
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Description
Test your knowledge on gene therapy, genetic disorders, and related concepts. This quiz covers various topics including titer, carrier vectors, and specific genetic conditions like ATTRv. Enhance your understanding of the interplay between genetics and therapy.
