Micro 9: Viral Vectors & Gene Therapy – Flashcards
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what is gene therapy?
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novel approach to treating disease based on modifying expression of a person's genes toward therapeutic goal
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first clinical trial for gene therapy? year, strategy, disorder
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1990, ex vivo, Adenosine Deaminase Deficiency
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what was the first in vivo gene therapy treating? what did it use?
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cystic fibrosis treated with attenuated adenovirus
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t/f gene therapy is highly experimental collection of technologies; lots of potential
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true
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what does somatic gene therapy involve?
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manipulation of gene expression in cells corrective for patient; will NOT be passed on to further generations
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what is germline gene therapy?
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involves genetic modification of germ cells; these WILL change the next generation
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which type of therapy somatic/germline, is limited to animal models?
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germline
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steps in ex vivo gene therapy treatment of Familial Hypercholesterolemia
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1. remove pt liver, treat cells with retrovirus LDL receptor gene 2. discard liver cells that did not take up corrective genes 3. reimplant corrected genes into patient liver
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what are the 4 main disease states being targeted for gene therapy intervention?
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1 Cancer 2 Monogenic diseases 3 Adenosine deaminase deficiency (SCID) 4 Leber congenital amaurosis
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what is a vector?
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modified, attenuated viruses that are used to deliver payload(gene) into a cell
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what is a therapeutic strategy?
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vector carries a gene that encodes a protein that is defective/not present in host genes
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what is a cytolytic strategy?
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vector is designed to kill or eliminate diseased cell/tissue
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example of cytolytic therapy; what converts prodrug glancyclovir to toxic drug?
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expression of thymidine kinase (TK)
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What is CAR-T Cell Immunotherapy?
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Chimeric Antigen Receptor on a patients Tcell; programming patients own cells to attack cancer
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Kymriah specifically recognizes what Bcell marker?
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CD19
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with CAR-T Cell Immunotherapy for Acute Lymphoblastic Leukemia: how are Tcells collected? what are they infected with? how are Tcells reintroduced?
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-collected by apheresis -infected by viral vector with CAR -the Tcells are reintroduced via Tcell adoptive transfer
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what group is specifically intended to use Kymriah?
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patients with Bcell ALL; intended for patients whose cancer has not responded to treatment
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where is CD19 found?
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surface of differentiated Bcells, NOT hematopoietic stem cells or other essentials
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how is CD19 utilized in treatment?
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CAR binds leukemic Bcells with CD19, then activates Tcells to destroy leukemic Bcells
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first step in production of CAR?
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In recognition domain, variable region gene segments cloned to construct a single-chain variable fragment
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what is the second step of CAR production?
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scFv is fused to extracellular CD8 domain and cytoplasmic CD3
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what accompanies scFV fusion to CD8?
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fuse to transmembrane/ cytoplasmic domain of CD3
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how do you get CAR into patient's Tcells?
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retrovirus, viral vector used to transduce Tcells ex vivo
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why are retroviruses used to integrate patient's Tcells?
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CAR gene will integrate into genome of Tcell
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what are the steps involved in development of a genetic therapy?
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1 identify a gene responsible for dz; clone functional cDNA 2 delivery 3 control gene expression 4 avoid host immune response
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what is the goal of delivery?
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get genetic material to the appropriate cell types: specific, efficient, safe
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what are most vectors based on?
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attenuated or modified versions of viruses
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what is the challenging part of delivery?
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remove disease causing components of virus and insert functional clones to treat
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what is involved in controlled gene expression?
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-making the correct amount of therapy protein, @ right time -maintain longterm expression
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when avoiding host immune response, what kind of vectors should be used?
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vectors that give minimal/no adverse immune response
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5 types of vectors
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adenovirus adeno-associated virus herpes virus liposomes/naked DNA retrovirus
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what is a retrovirus?
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enveloped virus with RNA genome; upon infection of cell converted to dsDNA by enzyme reverse transcriptase
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what allows for long-term expression of therapeutic drug during cell division?
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viral dsDNA insertion into genome
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how do they generate a gene therapy vector with retroviruses?
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viral genes encoding structural proteins are deleted and replaced with therapeutic gene of interest
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what is a disadvantage of murine-based retrovirus vectors?
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require replicating cells for genome integration
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what is a Lentivirus? what is the advantage of using it as a vector?
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HIV-based gene vector that can integrate into both replicating and non-replicating cells
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what is an adenovirus?
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non-enveloped virus with large dsDNA genome that accommodates large inserts
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how does an adenovirus enter cell?
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binds to specific receptors, delivered to nucleus following uncoating; replicates episomally
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which proteins are deleted for adenoviruses?
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structural proteins and proteins that induce cell division
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what is a liposome vector?
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synthetic lipid that binds to and encapsulates plasmid DNA; fuse with cell membrane and deliver genetic payload
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why is it that liposomes can deliver to many cell types?
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do not bind to specific cell receptors
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why are structural proteins removed when making a vector?
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eliminates the possibility that the vector can become a self-replicating virus
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first step in making a retroviral vector ends in "packaging cell line"; what is the process?
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-genes encoding structural proteins deleted from genome and cloned into plasmid expression vector -plasmid is then transfected into cell; express structural proteins not viral genomes (done in cell culture)
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what is the second step in making a retroviral gene therapy vector?
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sequences from virus that result in packaging of genetic material into virus (aka packaging signals) are removed, put in plasmid -therapeutic gene of interest is cloned between packaging signals
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what is the third step in retroviral vectors ending in the "producer cell line"?
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plasmid with packaging signals introduced into packaging cell line, structural proteins assemble therapeutic gene into virus particles
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what is the fourth step in making retroviral vectors?
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virus released from packaging cell to infect new cells; only delivers therapeutic gene into cells
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first problem with gene therapy that needs to be solved?
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how to avoid an immune response; inflammation or antibodies
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second problem with gene therapy that needs to be solved?
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how to get genes efficiently into non-dividing cells (liver, muscle, neurons)
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what is the third problem associated with gene therapy that needs to be solved?
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how to get genes integrated so that they will not be deleted or over-expressed
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what is adeno-associated virus (AAV)?
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small virus which infects humans and some other primate species
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how did AAV get its name? why is it advantageous as a gene therapy vector
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found in cells simultaneously infected with adenovirus; by itself it is harmless
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how does AAV differ from adenovirus? (3)
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-does not stimulate inflammation in the host -does not elicit antibodies against self -can integrate on chr19, but inefficiently
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what is Glybera?
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AAV1 carrying lipoprotein lipase gene
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with LPL deficiency patient cannot break down _______; the clinical presentation is (4)
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pt cannot break down fats; - abdominal pain -acute and recurrent pancreatitis -eruptive cutaneous xanthoma -hepatosplenomegaly
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Success with gene therapy for SCID-X1 disease: deficiency? result? what vector was used?
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-deficiency of gamma-subunit for IL-2, 4, 7, 9, 15 -causes block T and NK cell differentiation -retrovirus vector encoding gamma-subunit and exvivo CD34+ cells
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what is the success rate of gene therapy for SCID-X1?
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10/11 infants cured
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Adenosine deaminase deficiency SCID? deficiency? result? vector used?
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-purine metabolism defect -impaired lymphocyte development and function -retrovirus vector containing ADA cDNA and exvivo CD34+ cells
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with correction of ADA-SCID, what did patients undergo before receiving gene therapy?
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patients underwent mild nonmyeloablative condtioning with busulfan
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what is the success rate of gene therapy for ADA-SCID?
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both patients treated in this specific study are home and well
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Leber congenital amaurosis cause? result? vector used?
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-bialleic mutations in RPE65 gene -blinding retinal diseases -rAAV2 carrying human RPE65 gene
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what was the outcome of LCA treatment with rAAV2?
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all 12 patients responded well; 6 restored vision, not legally blind
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who was the first death directly attributed to gene therapy? what did the patient have? what treatment did the patient receive?
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Jesse Gelsinger had ornithine transcrabamylase (OTC) deficiency; he was infused with an adenovirus derived vector directly into hepatic artery
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what was the suggested cause of Jesse Gelsinger's death?
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multiorgan failure from anoxia; levels of cytokines suggest systemic inflammatory response syndrome(SIRS)
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how long after XSCID study did patients begin to develop leukemia?
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3 years after treatment
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strong evidence leads investigators to believe leukemia is due to "___________ _________________"
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insertional oncogenesis
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why is "insertional oncogenesis" considered an unlikely but possible risk for this gene therapy?
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vectors cannot reproduce themselves and so cannot repeatedly insert into cell's chromosomes...
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what is Clustered Regularly Interspaced Short Palindromic Repeats CRISPR/Cas9?
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segments of bacterial DNA containing short, repetitive base sequences that form hairpin structures
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the _____________ sequence precedes the hairpin; it confers sequence _______________
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target sequence sequence specificity
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what makes up the Guide RNA?
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target sequence + hairpin structure
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what is a Cas protein? how does it work with target sequence?
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Cas proteins are endonucleases; together with target sequence they recognize and cut exogenous DNA
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what does the CRSPR/Cas9 system have the capability to do
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edit genomic DNA of any species by delivering Cas9 nuclease+ synthetic RNA into cell
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what are 5 factors influencing development of gene therapy??
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1 optimize vector safety 2 overcome tech obstacles 3 analysis of human genome 4 expansion of diagnosis industry 5 public acceptance of gene therapy
