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3 uses for gene therapy

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- Correct defective genes - Treat cancer - Deliver DNA vaccines

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Most common approach for gene therapy

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Gene inserted into a nonspecific location within the genome

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Main differences between non-DNA and DNA immunization

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Non-DNA: Usually live attenuated or inactivated, usually contaminated and can degrade because they are proteins. Require refrigeration. DNA: MAY give potent immune response since antigens are expressed over a long period (once incorporated/expressed). Usually more stable, no refrigeration required.

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DNA vaccine administration

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- Injection (intramuscular- use linear or circular DNA?) - Use gene gun (DNA coated on particles) - Electroporator (use electricity to get DNA into cells) - Topical application (DNA in gel/cream applied to skin) - Promoters?

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Gene therapy steps

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1. Identify gene(s) responsible for disorder 2. Make copies of normal gene 3. Insert copies into vectors (viruses) - Remove viral genome - Insert gene of interest 4. "Infect" affected cells with vectors - Normal gene carried into nucleus - DNA may integrate into genome 5. Activate the gene - Transcription and translation take place

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Advantages of using liposomes in gene therapy

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- Stable complex - Can carry large sized DNA - Can target specific cells - Does not induce immunological reactions

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Disadvantages of using liposomes in gene therapy

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- Low transfection efficiency - Transient expression - Inhibited by serum - Some cell toxicity

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CFTR

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Cystic fibrosis transmembrane conductance regulator

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Describe two types of CFTR channels (normal an mutant)

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Normal: moves chloride ions to outside of cell Mutant: does not move chloride ions, causing sticky mucus to build up outside of the cell

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LPN

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Lipopeptide Nanoparticles Used for potent and selective siRNA delivery in rodents and nonhuman primates

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Draw a table of the steps of gene therapy beginning with exogenous DNA including the 3 final outcomes

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(see screenshot on desktop)

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List the barriers to successful gene therapy

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1) Vector development 2) Corrective gene construct 3) Target cell proliferation/maintenance 4) Efficient transfection and transport of DNA to nucleus for genome integration 5) Expansion of engineered cells, implantation into patient

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List and describe the 3 categories of somatic cell gene therapy

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- Ex vivo: Cells removed from body, incubated w/vector and engineered cells returned to body. Blood cells often used. - In situ: Vector placed directly into affected tissues - In vivo: Vector injected directly into person, often blood stream

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List the in-vivo gene therapy approaches

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Intratumour Intravenous Intraperitoneal Subcutaneous Intramuscular Intra-arterial Airways Lung

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List the steps involved in ex-vivo gene-therapy approaches

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1) Biopsy cells retrieved from patient (bone marrow, dermis, liver, tumour) 2) Grow cells 3) Transduce cells via retrovirus or DNA vector 4) Quality control 5) Return to body

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Why are viruses ideal vectors for gene therapy?

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- Have evolved to infect cells w/specificity - Efficiency re: transfecting own DNA into host cell genome allowing production of new viral particles at period of synthesis of the cell

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List the ideal viral vector characteristics

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1) Insert size: 1 or more genes 2) Targeted: limited to cell type 3) Immune response: none 4) Stable: not mutated 5) Production: easy to produce high concentrations 6) Regulatable: produce enough protein to make difference 7) Infect dividing and non-dividing cells 8) Integration

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What are the two types of viral vectors?

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RNA viruses (retroviruses) and DNA viruses

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List the most prominent retroviruses used in gene therapy

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MuLV: Murine leukaemia virus HIV: Human immunodeficiency viruses HTLV: Human T-cell lympotropic viruses

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List the most prominent DNA viruses used in gene therapy

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Adenoviruses AAV: Adeno-associated viruses HSV: Herpes simplex virus Pox viruses Foamy viruses

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MuLV

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Murine leukaemia virus Prominent RNA virus (retrovirus) used as a viral vector in gene therapy

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HIV

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Human immunodeficiency virus Prominent RNA virus (retrovirus) used as a viral vector in gene therapy

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HTLV

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Human T-cell lymphotropic viruses Prominent RNA virus (retrovirus) used as a viral vector in gene therapy

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Adenovirus

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Prominent DNA virus used as a viral vector in gene therapy

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AAV

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Adeno-associated viruses Prominent DNA virus used as a viral vector in gene therapy

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HSV

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Herpes simplex virus Prominent DNA virus used as a viral vector in gene therapy

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Pox virus

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Prominent DNA virus used as a viral vector in gene therapy

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Foamy virus

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Prominent DNA virus used as a viral vector in gene therapy

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List examples of gene therapy trials

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- Adnosine deaminase gene transfer to treat severe combined Immuno-Deficiency (SCID) - CFTR gene transfer to treat Cystic Fibrosis - Advanced Central Nervous System Malignancy - Mesothelioma - Ornitine Transcarbamylase deficiency - Hemophilia - Sickle cell disease

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SCID

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Severe Combined Immuno-Deficiency "Baby/boy in bubble syndrome" SCID-X1 most common; defect in cytokine cell-surface receptor. Failure to develop mature T-, B- and NK cells

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Draw a diagram of a retrovirus

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(see screenshot on desktop) Components: Lipid envelope, receptor binding proteins, RNAse, viral RNA, capsid: core proteins

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Advantages of using RNA viruses (retroviruses) in gene therapy

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- Randomly integrates into genome - Wide host range - Long term expression of transgene

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Disadvantages of using RNA viruses (retroviruses) in gene therapy

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- Small carrying capacity for therapeutic genes - Infectivity limited to dividing cells - Inactivated by complement cascade - Safety - Do not target specific cells - Randomly integrate into genome

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Draw the retrovirus vector construction for gene therapy

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[5' LTR][Packaging][Gene X][Neo^r][3'LTR]

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LTR

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Long terminal repeats in gene sequencing

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Steps of RNA virus gene therapy

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1) Packaging cell line (293s) + vector (transfection) 2) Collect virus after 24-72 hours 3) Concentrate virus/high titer 4) Transduce host cell

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Transfection

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Process of deliberately introducing nucleic acids into cells.

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Transduction

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Transfer of genetic material from one organism (as a bacterium) to another by a genetic vector

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List steps for creation of retroviral vectors

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1) Delete gag, pol, env genes. This is viral vector 2) Insert gene needed for therapy (gene X) into viral vector 3) Insert viral vector containing gene X into packaging cell line containing retroviral gag, pol and env genes

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ADA deficiency

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Adenosine deaminase deficiency First gene therapy trial Body needs ADA to convert deoxyadenosine to non-toxic substance

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Deoxyadenosine

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Intermediate product made during break-down and synthesis of DNA. Natural in body. Toxic when not converted to non-toxic form by ADA.

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Describe Fischer's X-Linked SCID clinical trial and its' results

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- 11 patients 1-9mo - Bone marrow aspirate; selection of CD34+ progenitors - 3 daily exposures to retroviral vector carrying yc chain cDNA - Reinfusion of marrow - 2 developped leukaemia due to insertion near LMO-2 gene; all T cells were eventually clones of one cell within LMO-2 gene

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CD34+

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Progenitors selected for during X-linked SCID clinical trial

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LMO-2

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Gene associated w/leukaemia

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Draw a diagram of an adenoviruss

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(see screenshot on desktop) Includes fibre, viral DNA, capsid, knob

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Advantages of using adenoviruses in gene therapy

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1) High transduction efficiency including non-dividing 2) High gene expression level 3) Slightly increased capacity for exogenous DNA 4) Large amounts easily produced

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Disadvantages of using adenoviruses in gene therapy

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1) Potentially transient expression 2) Cell-specific targeting difficult to achieve 3) Ubiquitous virus uptake 4) Safety 5) Removal of E3 region causes virus to be more susceptible to immune system 6) Transfected cells often targeted by immune system

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OTCD

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Ornithine Transcarboxylase Deficiency Live enzyme deficiency disease carried by Jesse Gelsinger, first patient to die from gene therapy

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Jesse Gelsinger

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First patient to die from gene therapy; Body launched raging attack on inserted adenovirus resulting in brain death. High ammonia levels blamed; university failed to report two patients w/serious side effects; deaths of monkeys not included in informed consent discussion

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Describe the two host immune activation complications of gene therapy

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1) Innate immune responses cytokine elevations -> acute systemic illness/death 2) Cell mediated/humoral responses vector -> cytotoxicity/re-administration futile and/or transgene product -> antibody development

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Gene therapy risks

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- Viruses affecting many types of cells - Immune reaction - Viruses transmitted to others, causing infection - Insertional mutagenesis - Viral DNA may be introduced into germ cells producing inheritable changes

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Insertional mutagenesis

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Gene inserted in wrong location during gene therapy causing cancer

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List steps of gene therapy as a cancer treatment

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1) Insert functional copy of gene for inactive/defective one; cancer cells now sensitive to anticancer drugs 2) Insert multidrug resistant gene (MDR) into stem cells making them more resistant to side effects of anticancer drugs

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MDR

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Multidrug resistant gene Making stem cells more resistant to side effects of anti-cancer drugs

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List steps of ES isolation

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1) Sperm and egg join, zygote formed 2) 5-7 day development; Blastocyst 3) Remove inner cell mass 4) Grow in dish 5) Change culture conditions to stimulate cells into variety of cell types 6) Results: Skin/skeletal/muscle/neural cells

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List steps of creating ES cells through therapeutic cloning

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1) Isolate cells from patient 2) Remove nucleus from an egg cell 3) Transfer nucleus from patient's cell to egg 4) Egg cell "reprograms" to patient's DNA 5) Stimulate cell to begin dividing; let develop to blastocyst stage 6) Isolate inner cell mass from blastocyst and grow in dish

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List steps of creating iPS cells

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1) Isolate skin or fibroblast cells from patient, grow in dish 2) Treat cells w/reprogramming factors 3) Wait a few weeks 4) Pluripotent stem cells 5) Change culture conditions to stimulate cells to differentiate

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iPS cells

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Induced pluripotent stem cells Type of pluripotent stem cell that can be generated directly from adult cells.

29-30- Gene Therapy – Flashcards

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