BIOL 432 Test 1 Study Questions PDF

BIOL 432: Test 1 Study Questions Unit 1: CRISPR and Gibson Assembly 1. Describe the native function of the CRISPR/Cas9 system. (5 marks) a. CRISPR naturally functions almost like an immune system in bacteria that possess the system. b. CRISPR involves a CRISPR ar...

BIOL 432: Test 1 Study Questions Unit 1: CRISPR and Gibson Assembly 1. Describe the native function of the CRISPR/Cas9 system. (5 marks) a. CRISPR naturally functions almost like an immune system in bacteria that possess the system. b. CRISPR involves a CRISPR array, and CRISPR-associated proteins, such as Cas9. The CRISPR array is composed of variable spacer regions, separated by repeat regions. The variable spacer regions are actually derived from past bacteriophage infections. c. When a bacteriophage infection occurs, the CRISPR array is transcribed, generating crRNA fragments of the variable spacer regions. d. If this bacteriophage has infected this bacterial lineage before, one of the crRNAs will be complementary to the bacteriophage genome. The crRNA will associate with Cas9 nuclease, and guide it to the complementary spot on the bacteriophage genome. Cas9 will cleave the bacteriophage genome in close proximity to where the crRNA anneals, and stop the infection. e. If this bacteriophage has not infected this bacterial lineage before, Cas9 will cleave at PAM sites, without crRNA binding, to generate a fragment of the genome that can be incorporated into the CRISPR array, to protect against a future infection. 2. Describe the biotechnological use of the CRISPR/Cas9 system. (6 marks) a. We can introduce the Cas9 enzyme and a gRNA specific to the site we want to edit. For example, we might transform or transfect the target organism or tissue with an expression vector carrying the gene for Cas9 and the gene for the gRNA. b. Once inside the system, the gRNA will associate with the Cas9 nuclease. Cas9 nuclease will begin to scan the genome for PAM sites. At each PAM site, the gRNA will either be complementary and anneal, or not. c. If the gRNA is complementary, each nuclease domain of Cas9 introduces a nick. The result is a double stranded break upstream of the PAM site. d. The cell will attempt to repair the dsDNA break. Some organisms preferentially perform homology directed repair (HDR), while others preferentially perform non-homologous end joining (NHEJ). e. When NHEJ occurs, the cell will join two non-homologous ends of DNA. Some insertions and deletions occur, usually resulting in a knockout of whichever gene the break occurred in. f. When HDR occurs, the cell will use a homologous sequence to repair the break. In this case, we supply “donor DNA” which contains regions that are homologous to regions flanking the break. The donor DNA might carry a point mutation or a whole gene that we wish to introduce. When the donor DNA is used to repair the break, our intended mutation is also incorporated. 3. What is a PAM site? What is its function? Where is it? (3 marks) a. A PAM site is the NGG motif that Cas9 nuclease binds to as the first step in potentially cleaving DNA.  b. The PAM site is on the strand opposite the strand that the guide RNA binds to. 4. What are the two components of guide RNA in a native CRISPR system? (2 marks) a. Guide RNA in a bacterial cell that uses CRISPR is composed of crispr RNA (crRNA) and tracrRNA (trRNA). crRNA is complementary to the target sequence and provides specificity to Cas9 cleavage. trRNA anneals to crRNA and binds to Cas9, thus attaching the guide RNA to the Cas9 enzyme. 5. What is the major drawback to CRISPR? (1 mark) a. Off-targeting is the major drawback to CRISPR. Off-targeting is when Cas9 cleaves the genome somewhere that is not the target location. 6. What is multiplexing? (1 mark) a. Multiplexing is the introduction of multiple gRNAs at the same time, during a CRISPR experiment. For example, you could introduce one gRNA to delete a gene on one chromosome, and another gRNA to introduce a point mutation on another chromosome. The result is multiple mutations per one iteration of CRISPR. 7. Describe two other techniques for gene-editing, besides CRISPR. (4 marks) a. Zinc finger gene editing uses an enzyme with zinc-finger motifs specific to your target DNA sequence, linked to a FokI nuclease. To use ZFNs, you must design two enzymes, specific to the same location on complementary strands of DNA. When the enzymes bind, the FokI nucleases will be in close proximity to one another, and can introduce a dsDNA break. The dsDNA break may be repaired via NHEJ or HDR, as with CRISPR. b. TALENs gene editing uses TALE enzymes designed to bind to target regions of DNA, and a FokI nuclease. Again, you must design two TALENs, one for each strand of DNA, such that the FokI nucleases will be in close proximity to one another and can introduce a dsDNA break. 8. Why is CRISPR advantageous over using ZFNs or TALENs? (2 marks) a. CRISPR is advantageous because it requires selection of a gRNA specific to the target location, while ZFNs and TALENs both require the design and synthesis of an entire enzyme specific to the target location. Thus, CRISPR is simpler, quicker, and cheaper. 9. Suppose we want to introduce a point mutation into chromosomal DNA using CRISPR. What do we need? What is the purpose of each? (4 marks) a. Cas9 nuclease → performs DNA cleavage to facilitate repair b. Guide RNA → guides Cas9 to the target location c. PAM site → the binding site for Cas9 in DNA, must be in close proximity downstream of the gRNA binding site d. Donor DNA → must include flanking homologous regions to facilitate HDR, also includes the point mutation we wish to incorporate 10. Briefly describe two modified versions of Cas9 and a biotechnological use for each (4 marks). a. dCas9 → Cas9 with both nuclease domains deactivated, such that it does not cleave DNA  i. Can be used to down-regulate gene expression by using a gRNA to target it to bind to a gene, thereby blocking binding of transcription machinery. ii. Can be used to down-regulate gene expression by attaching a transcriptional repressor, and directing dCas9 to bind in close proximity to a promoter we wish to suppress. iii. Can be used to up-regulate gene expression by attaching a transcriptional activator, and directing dCas9 to bind in close proximity to a promoter we wish to induce. iv. Can be used to visualize elements of DNA, by attaching a marker (eg. GFP) to dCas9. v. Can be used to introduce site-specific mutations without DNA cleavage, by attaching a deaminase domain. Deaminase activity can convert cytosine to thymidine, which can be used to fix deleterious mutations or introduce stop codons. vi. We can add a FokI domain to dCas9, and use two gRNAs to target dCas9 to regions in close proximity to one another. When the FokI domains are in the same position on opposite strands, they will introduce a dsDNA break. b. Cas9 nickase → Cas9 with one nuclease domain deactivated, such that it introduces nicks (ssDNA breaks) i. A pair of complementary nickases can be used together, with gRNAs to target opposite strands in close proximity to one another, to introduce two nicks close together. These nicks behave like a dsDNA break, and can induce NHEJ of HDR. This is a strategy to reduce off-target mutations, because the Cas9 nickases must cleave in close proximity for a break to occur, and off-targeting of either nickase individual will generate a nick that is easily repaired. 11. How can we alter gRNA to increase the specificity of CRISPR cleavage? (2 marks) a. We can alter the gRNA so it is 20 nt long and includes a GG motif on the 5’ end. b. We can alter the gRNA so it is 17 nt long. 12. What is CRISPR 2.0? (2 marks) a. CRISPR 2.0 is a CRISPR system that uses Cpf1 as the nuclease. Cpf1 introduces staggered cuts (similar to restriction enzyme digestion) and is more specific than Cas9. 13. What are biohackers? (1 mark) a. Biohackers are people who take advantage of biotechnology in their personal lives, outside of organized research. 14. What is Gibson assembly? Describe the steps of the reaction. (4 marks) a. Gibson assembly is a technique for the scarless assembly of multiple DNA fragments within a single-tube reaction. b. First, the fragments we wish to assemble must be amplified with PCR primers that will add homologous sequence to either end of the fragments. For example, if we wish to assemble fragments A, B, and C in that order, we would use a reverse primer for fragment A with a 5’ region that is complementary to the 5’  region of the forward primer for fragment B. Similarly, we would use a reverse primer for fragment B with a 5’ region that is complementary to the 5’ region on the forward primer for fragment C. c. Next, we combine the amplified fragments with the Gibson assembly master mix, which contains 5’ exonuclease, DNA polymerase, and DNA ligase. d. In the reaction, the exonuclease will “chew back” the 5’ strand to generate 3’ overhangs on each fragment. Complementary overhangs can anneal, then DNA polymerase fills in the gaps to generate one fragment with nicks. Finally, DNA ligase seals the nicks to generate one dsDNA molecule of the combined fragments. Unit 2: Protein Therapeutics 15. What is a protein therapeutic? Name one example. (2 marks) a. A protein therapeutic describes a protein that is administered to treat a diseased state. i. For example, Herceptin is a monoclonal antibody used to treat breast cancer. ii. For example, Humira is a monoclonal antibody used to treat rheumatoid arthritis. iii. For example, Rituxan is a monoclonal antibody used to treat non-Hodgkin’s lymphoma. 16. What is the largest category of protein therapeutics? (1 mark) a. Antibodies 17. Briefly describe the steps between idea and commercialization phases of development of a drug. (10 marks) a. The discovery phase involves learning about the drug, prior to testing on humans. i. Often involves the initial screening of millions of compounds for a target activity. ii. Then, the preclinical pharmacology phase focuses on extensive laboratory research on mode of action, chemical structure, and biochemical and physical properties of the potential drug. iii. Then, the preclinical safety phase focuses on assessing the harm of different doses to small animals (eg. mice) iv. Then, the clinical pharmacology and safety phase involves assessing the harm of different doses on higher animals. b. The exploratory development and full development phases involve phase I, II, and III clinical trials on humans. i. Phase I clinical trials test safety of the drug by assessing the highest dosage that can be administered to healthy individuals without serious side effects. ii. Phase II clinical trials test appropriate dosage by using test and control groups to determine the optimal dosing regime.  iii. Phase III clinical trials test the magnitude of the effect of the drug on the disease phenotype. c. After clinical trials, commercialization begins and the product can be released to market. 18. How is recombinant DNA technology used to produce protein therapeutics? What are the advantages of this? (5 marks) a. Recombinant DNA technology can be used to produce protein therapeutics by genetically modifying expression systems to produce our protein of interest in large quantities. b. The advantages to this are: i. We can produce the therapeutic in large quantities ii. It is cheaper iii. It allows us to easily create variants of the therapeutic iv. The process is highly reproducible 19. Describe how recombinant technology was used to produce interferon therapeutics. (4 marks) a. Interferon is naturally expressed in animal cells, in response to viral infection. When a viral infection occurs in one cell, interferon is produced and secreted. It then interacts with receptors on neighbouring cells and induces antiviral protein production. b. Interferon can also be administered as a protein therapeutic against viral infections. Recombinant technology was used to generate a hybrid interferon from fragments of several natural interferons, to increase its efficacy as a therapeutic. i. Specifically, using restriction enzymes to generate fragments of several interferon genes, then re-ligating fragments together to create combinations of the fragments. ii. Or, use PCR synthesis to assemble fragments in a specific order, using a set of overlapping primers. 20. What is the half-life of a drug? Describe three ways to improve the half-life of drugs (4 marks). a. The half-life of a drug is the time it takes for half of the initial dose to be removed from the body. b. Pegylation (ie. the attachment of polyethylene glycol (PEG) to other molecules) can be used to decrease renal clearance, and thus increase half-life of therapeutics. c. We can also use dimerization of therapeutics to increase half-life. For some therapeutics, dimerization can be used to decrease the rate of degradation, and thus increase the half-life. d. We can also increase the half-life of proteins by fusing an XTEN domain, which increases stability, solubility, and resistance to aggregation, which then increases half life. 21. How is increasing the half-life of therapeutics beneficial to the patient? (2 mark)  a. Increasing the half-life means the therapeutic persists in the bloodstream for longer. This means we can dose less of the therapeutic at a time, or dose less frequently, which usually decreases the cost of the therapy to patients. 22. Describe one example of a non-antibody protein therapeutic (2 marks). a. The use of DNase and alginate lyase to treat cystic fibrosis i. Delivery of modified DNAseI (that does not bind actin) via an inhaled mist → degrades DNA and thins mucus in the lungs ii. Delivery of modified alginate lyase (mutated to remove antigenic epitopes) via an expression vector to degrade alginate produced by P. aeruginosa during infection and reduce biofilm formation in the lungs b. Bacteriophages to treat cystic fibrosis i. Administer a non-lytic bacteriophage carrying an adjuvant gene (eg. M13 carrying LexA) → when the gene is integrated into the bacterial genome and expressed, LexA acts as an adjuvant that makes the cells sensitive to quinolone antibiotics c. Overexpression of IL-10 by LAB to treat Crohn’s disease and ulcerative colitis i. Engineer LAB to over express IL-10, which modulates T-cells, and administer them to the patient ii. These LAB are also designed to lack an essential gene (eg. thymidylate synthase), such that they cannot proliferate outside of the human body d. Treatment of obesity in mice with leptin production by LAB i. Engineer LAB to secrete leptin, and introduce them into obese patients → when expressed, leptin tells the body it is not hungry anymore, and can be used to help diet/weight loss regimes e. Expression of cyanovirin N by LAB to prevent the transmission of AIDS i. Cyanovirin N is a protein that binds gp120 and gp41 entry proteins on the AIDS virus, and prevent entry into host cells ii. Introduction of cyanovirin N overexpressing LAB can be used to reduce transmittance of the AIDS virus f. Production of bioidentical insulin by LAB i. Engineer LAB to express bioidentical insulin, and administer to patients with diabetes 23. Briefly describe the steps of immunogenic recognitions of foreign entities (6 marks). a. A macrophage takes in proteins from the extracellular environment via phagocytosis b. Within the endosome, the proteins are processed via proteolysis, producing fragments (ie. epitopes) c. Protein fragments bind MHCs, and then the antigen-bound MHC is transported from the endosome membrane to the cell membrane d. The MHC complex holds the epitope facing the extracellular environment, for recognition by T-cells e. If/when a helper T cell binds the epitope, it undergoes a change in gene expression and begins secreting cytokines and differentiation  f. The production of more helper T cells facilitates an immune response against the recognized epitope 24. What is a hybridoma? (1 mark) a. A hybridoma is a fusion between a plasma cell with a myeloma cell that can produce antibodies and is immortal. 25. Describe the events of monoclonal antibody production (5 marks). a. Inoculate a mouse, rat, or rabbit with the antigen we wish to generate mAb against b. Euthanize the animal, harvest the spleen, and isolate B-cells c. Combine B-cells, HGPRT- myeloma cells, and polyethylene glycol (PEG) to facilitate fusion d. Plate the mixture on HAT media, which only allows hybridomas to grow (functional complementation) e. Screen hybridomas to ensure they are producing antibodies against the initial antigen by diluting into multi-well culture plates and testing supernatant via ELISA 26. Describe hybridoma selection using HAT media (5 marks). a. To generate hybridomas, mix B-cells, myeloma cells and PEG to facilitate membrane fusion. The result is a mixture of cells and hybrids. There may be unfused B cells, fused B cells, unfused myeloma cells, fused myeloma cells, and fused B-cell myeloma cell hybrids (ie. hybridomas). b. Plate this entire mixture on HAT media to select for hybridomas only. HAT media selects for hybridomas because it contains aminopterin, which inhibits de novo dGTP and dTTP synthesis. It also contains thymidine and hypoxanthine, which enable dGTP and dTTP synthesis via the salvage pathway, which requires HGPRT-. c. B-cells are HGPRT+, so unfused and fused B-cells can undergo the salvage pathway and proliferate, but they are not immortal so they will die quickly. d. Myeloma cells are HGPRT-, so unfused and fused myeloma cells will not be able to synthesize dGTP or dTTP, and thus cannot proliferate. e. Hybridomas are HGPRT+ (conferred by the B-cell portion) and are immortal (conferred by the myeloma portion), thus they can synthesize dGTP and dTTP and proliferate for an extended period of time. 27. Describe two advantages of using hybridomas for monoclonal antibody production (2 marks). a. Hybridomas are immortal, thus there is no need to regenerate another monoclonal antibody against the antigen, thus the antibody is identical every time b. Hybridomas are a long-term supply of monoclonal antibodies because they can be cryopreserved and thawed as needed. 28. What is the drawback to using non-human antibodies as a therapeutic in humans? (1 mark) a. Antibodies generated in another organism will possess a species-specific Fc region which will generate an immune response in humans, resulting in destruction of the antibody. 29. Describe the two types of humanized antibodies (4 marks).  a. Chimeric (aka. Partially humanized) antibodies contain the Fc region of humans fused to the antigen-specific Fv region from another organism (eg. mice) that was used to generate antigen-specific monoclonal antibodies. b. Humanized (aka. 95% humanized) antibodies contain the Fc region from humans and the Fv region of humans that also includes CDRs specific to the target antigen, from a non-human organism. 30. What are CDRs? (1 mark) a. Complementarity determining regions (CDRs) are hypervariable domains within the Fv region of an antibody that determine epitope specificity and binding of the antibody. 31. Suppose you identify antigen-specific CDRs in a monoclonal mouse antibody, and you want to make a 95% humanized antibody against that antigen. What technique could you use to generate this antibody? (1 mark) a. You could use gibson assembly to assemble CDRs into the Fv region of a human antibody to generate a 95% humanized antibody. b. Alternatively, you could use PCR synthesis. 32. What are XenoMice? (1 mark) a. XenoMice are “humanized” mice that have been engineered to produce human antibodies. 33. Describe the process of generating XenoMice (4 marks). a. Delete the IgH and IgK genes (ie. genes for light and heavy chains) from mouse embryonic stem cells to generate an IgH- IgK- deficient mouse line b. Introduce human IgH and IgK genes into another mouse use a yeast artificial chromosome (YAC) c. Breed the two mice lines together and select for offspring that are deficient for mouse IgH and IgK and produce human IgH and IgK d. Immunize these mice with an antigen, harvest the spleen, and generate hybridomas → the hybridomas will produce human antibodies 34. What are antibody fragments? What is one advantage to using antibody fragments over whole antibodies? (2 marks) a. Antibody fragments are combinations of Fv and Fc antibody regions that are generally smaller than whole antibodies. b. The advantage to using antibody fragments over whole antibodies is that they are cheaper to produce and they infiltrate tumors easier because they are small. 35. What is a diabody? Briefly describe the two types of diabodies (2 marks). a. A diabody is an antibody fragment composed of two Fv domains, but no Fab or Fc regions. b. Bivalent diabodies are composed of two identical Fv domains, while bispecific diabodies are composed of two different Fv domains. 36. What is one drawback to the use of antibodies as a protein therapeutic? (1 mark) a. Antibodies have a relatively short half-life as they are degraded after ~9 days. 37. How can we increase the half-life of antibodies? (1 mark) a. We can perform random or directed mutagenesis on the Fc domain and assay for mutations that increase the half-life.  38. Briefly describe an example of an antibody used as a protein therapeutic (2 marks). a. The use of antibodies against C. diphtheriae to treat diphtheriae b. Rituximab is a monoclonal antibody against the CD20 antigen on the surface of B cells as a treatment for non-hodgkin's lymphoma. It kills B-cells by binding to them and inducing apoptosis, and by recruiting macrophages, monocytes, and natural killer cells. It targets cancerous and non-cancerous B cells, but does not kill stem cells, so healthy B-cells can regenerate after treatment. c. Herceptin is a monoclonal antibody against the HER2 receptor, which prevents the binding of growth factors and thus prevents uncontrolled growth. d. Select CDRs have been identified that efficiently target the Epstein-Barr virus. e. An antibody with two Fv regions (one specific to IL-12, one specific to IL-18) has been developed. f. To treat cancer, irinotecan is administered to induce expression of unique cell surface proteins on colorectal cancer cells. Then, a toxin-conjugated monoclonal antibody against those surface proteins is administered. Delivery of the toxin to the cancer cells causes cell death and decreased tumor size. g. Monoclonal antibodies against ROR1 receptors have been used to treat ovarian cancer. However, ROR1 is a tyrosine kinase, and antibody binding causes dimerization, which can have cancerous side effects. Monovalent antibodies (ie. with one Fab domain only) have been used to prevent this dimerization. h. The use of humanized antibodies against a surface protein of HSV, in addition to acyclovir (an antiviral) can be used as a protein therapeutic to treat HSV. i. Abzymes have been used to target and degrade ghrelin, which decreases hunger levels and helps with weight loss. 39. What are abzymes? (1 mark) a. Abzymes, or catalytic monoclonal antibodies, are antibodies with active sites that possess catalytic activity. Unit 3: Nucleic Acid Therapeutics 40. List and briefly describe the four categories of nucleic acid therapeutics (4 marks). a. Antisense oligonucleotides → 15-24 nt in length, can target and bind mRNAs to down regulate expression by inhibiting translation b. Aptamers → bind motifs on proteins, peptides, drugs, or other molecules c. Ribozymes and deoxyribozymes → nucleic acid molecules that can cleave other nucleic acid molecules d. RNAi → small interfering RNA molecules that direct sequence-specific degradation of target mRNAs 41. Briefly describe three important characteristics of antisense oligonucleotides (3 marks). a. They must be readily taken up by cells, specific to efficiently target the mRNA, and resistant to degradation by cellular nucleases. 42. What are second-generation and third-generation antisense oligonucleotides? (2 marks) a. Second generation antisense oligonucleotides have 2’ methoxyethyl groups on the nucleotides of their 5’ and 3’ ends, phosphorothioate linkages, and deoxynucleotides in their center.  b. Third generation antisense oligonucleotides are less toxic and have a longer half-life than first- and second-generation antisense oligonucleotides. 43. List the three general guidelines for targeting antisense oligonucleotides (3 marks). a. It is most effective to direct antisense oligonucleotides to 5’ and 3’ ends of mRNAs, intron-exon boundaries, or regions that are naturally double stranded. 44. Describe an example of an antisense oligonucleotide nucleic acid therapy (2 marks). a. Antisense oligonucleotides can down regulate expression of target genes by binding complementary mRNA, inhibiting translation. The antisense oligonucleotide may be transformed/transfected directly into the cell, or introduced on a vector. b. Antisense oligonucleotides have been used to suppress malignant gliomas by transfecting with a vector containing cDNA for antisense ILGF-1 into tumor-causing cells. c. Antisense oligonucleotides can also be used to prevent retrovirus infection by binding to DNA elements and preventing transcription and integration of the viral genome into the host cell genome. d. Antisense oligonucleotides have also been used to treat psoriasis, by targeting and reducing transcription of mRNA for IGF-1 receptors. 45. What is the advantage of using aptamers over antibodies? (1 mark) a. Depending how they are prepared, antibodies can induce immune responses that result in their destruction. In contrast, aptamers are nucleic-acid based and generally are not recognized or destroyed by the immune system. 46. Describe the steps of SELEX. What is it used for? (7 marks) a. SELEX is a procedure for the generation of aptamers against a specific target molecule in which a large number of aptamers are generated and those that best bind the target molecule are enriched via iterative rounds of selection. b. To start, many (~106) random DNA sequences are cloned into vectors between a 3’ binding site for reverse transcriptase and a 5’ annealing site for PCR primers. c. T7 RNA polymerase transcribes these sequences into RNA aptamer-candidates. d. The library of aptamer-candidates is incubated with the target molecule. e. Unbound aptamers are washed away f. Bound aptamers are dissociated from the target molecule, converted to cDNA by reverse transcriptase, and amplified via PCR g. The process is repeated iteratively until only 1-2 aptamers that bind the target molecule with high affinity remain. 47. What are two modifications for aptamers and what do they do? (2 marks) a. We can modify aptamers to increase stability and decrease sensitivity to nuclease degradation by adding 2’ methoxy groups to purine residues and capping the 3’ end with deoxythymidine. 48. What is the main limitation of aptamer-based therapies in vivo? Describe one approach to improve this. (2 marks) a. The main limitation to aptamer-based therapies is maintaining high enough bioavailability in vivo.  b. We can improve aptamer bioavailability using liposomes or pegylation to decrease renal clearance and thus increase half life. 49. Describe an example of using aptamers as a nucleic acid therapy (2 marks). a. Aptamers (eg. Pegaptanib) can be used to slow macular degeneration by binding and inhibiting one isoform of vascular endothelial growth factor (VEGF). VEGF normally promotes angiogenesis in retinal epithelial cells, but errors in this process result in scarring, which causes a loss of vision. b. Aptamers have been designed to distinguish between wildtype p53 and mutant R175H p53 using a modified version of SELEX in which the mutant protein was bound to magnetic beads and the wildtype protein was bound to agarose beads. In each iteration, aptamers that preferentially bound the mutant protein were selected and amplified, while those that preferentially bound the wildtype were removed. The resultant aptamer was used to inhibit tumor formation in mutant mice. c. An aptamer bound to an siRNA was used to deliver that siRNA to cancerous prostate cells. The aptamer was specific to prostate-specific membrane antigens (PSMAs), while the siRNA was specific to lamin A/C proteins. Down regulation of nuclear lamins increases the susceptibility of the cancer cells to treatment. 50. What are ribozymes and deoxyribozymes? (2 marks) a. Ribozymes are catalytic 40-50 nt RNA molecules that bind specifically and cleave other RNA molecules. Deoxyribozymes are the DNA equivalent. b. They each have catalytic and substrate-binding domains. We can change the sequence of the substrate binding domain to change the target of the ribo/deoxyribozyme. 51. What are the advantages to deoxyribozymes over ribozymes? (4 marks) a. Deoxyribozymes are more efficient at binding some RNA molecules than their ribozyme counterparts. b. They are easier to synthesize c. They have broad target recognition properties d. They have high catalytic turnover 52. What is one limitation of ribozymes and deoxyribozymes? (1 mark) a. Delivery of ribozymes and deoxyribozymes into cells is problematic. 53. Describe an example of the use of ribozymes or deoxyribozymes as nucleic acid therapeutics (2 marks). a. Ribozymes can be used to cleave viral RNA and inhibit the expression of viral genes. b. A DNAzyme against c-jun mRNA has been used to inhibit skin cancer proliferation and metastasis. C-jun protein is a transcription factor component, whose overexpression is linked to several cancers. Cleavage of the mRNA with the DNAzyme down-regulates expression. 54. What is RNAi? Describe the events of RNAi in a cell (5 mark). a. RNA interference (aka. RNAi, post-transcriptional gene silencing) is the degradation of specific mRNA, based on the binding of small interfering RNA, which ultimately reduces gene expression.  b. When dsRNA is introduced into a cell it is cleaved into small fragments (siRNA) by dicer. c. The antisense strand of siRNA is incorporated into the RISC complex, which includes the Argonaute protein d. The antisense siRNA guides the RISC complex to the target mRNA, and the complex cleaves it e. Cleavage of the target mRNA results in downregulation of expression by up to 90% 55. List the considerations for designing siRNA for RNAi in vivo (4 marks). a. Include one mis-matched nucleotide to generate a “bulge” when dsRNA is formed ⇒ prevents activation of interferon, which can cause unpleasant side effects in recipients b. Avoid the sequence UGGC ⇒ it has a toxic effect c. Blunt ended 27 or 29-mers are more effective than 21-mer shRNAs 56. Describe an example of RNAi as a nucleic acid therapeutic. (1 marks) a. siRNAs have been used to degrade mRNAs with longer CAG repeats as a treatment for Huntington’s disease, preventing aggregation and deposition of improperly folded proteins. b. Onpattro is an RNAi drug for the treatment of hATTR. 57. Name and briefly describe the five common viral vectors (5 marks). a. Gammaretrovirus → an enveloped retrovirus with a small genome size, capable of stable integration into the host genome i. Eg. Moloney murine virus b. Lentivirus → an enveloped retrovirus with a rounded-trapezoid capsid and a slightly larger genome size, capable of stable integration into the host genome, specifically targets non-dividing cells i. Eg. HIV-1 c. Adenovirus → a naked virus with a 20-sided symmetrical capsid of hexagonal capsid proteins and a large dsDNA genome, that infects a variety of dividing and non-dividing cells, and does not integrate into the host genome d. Adeno-associated virus → a non-pathogenic, naked virus with a very small ssDNA genome that requires co-infection with a helper virus in order to replicate, different AAV strains target different tissue types, capable of stable integration into the host genome e. HSV-1 → an enveloped virus with a very large dsDNA genome that specifically targets neurons but does not integrate into host-cell chromosomes 58. Describe the events of adenovirus or adeno-associated virus infection of a cell (5 marks) a. The knob structure on the end of fiber proteins that protrude from the capsid binds surface proteins on the host cell membrane b. The virion is enclosed in a clathrin-coated vesicle, which fuses with an endosome that delivers the virus to the nucleus via microtubules c. At the nuclear pore, the adenovirus is released and disassembled, and viral DNA enters the nucleus d. For an adenovirus, replication of the viral genome occurs in the nucleus  e. For an AAV, if a helper virus is present, AAV replicates and forms new viral particles, and if a helper virus is not present, AAV integrates into chromosome 19 of the host cell 59. Suppose you want to deliver a 54 kb nucleic acid therapeutic to neurons. What viral vector would be appropriate for this and why? (2 marks) a. HSV-I would be most appropriate because it has a very large genome, and thus is capable of carrying large inserts. Second, it is capable of targeting neurons specifically. 60. What are the advantages to using HSV-I as a viral vector? (2 marks) a. HSV-I can carry up to 150 kb of sequence, thus it is suitable for carrying multiple gene copies or multigene constructs b. HSV-I does not integrate into the host cell genome, thus it poses no threat of integrational mutagenesis or cancer 61. List and briefly describe three non-viral delivery systems for nucleic acid therapeutics (3 marks). a. Direct injection of nucleic acid therapeutics → injection (or microinjection) of a naked nucleic acid therapy directly into the recipient b. Injection of lipid-linked siRNA molecules → link the nucleic acid therapeutic to a lipid molecule to overcome disadvantages to injection of naked nucleic acids, or use a lipopeptide nanoparticle c. Delivery via non-pathogenic bacteria → use a non-pathogenic bacteria to express a nucleic acid therapeutic in vivo d. Delivery via bacterial minicells → the use of small bacteria-derived cells to deliver nucleic acids to cells e. Delivery via dendrimers → a nanoparticle with nucleic-acid binding sites f. Delivery via antibodies → link a nucleic acid therapeutic to an antibody to direct that therapeutic to a specific target cell g. Delivery via aptamers → link a nucleic acid therapeutic to an aptamer to direct that therapeutic to a specific target cell 62. What are the disadvantages to direct injection of nucleic acids as a delivery method? (4 marks) a. Naked nucleic acids are cleared from the body rapidly ⇒ short half life b. They are degraded by nucleases in the serum ⇒ short half life c. They lack organ-specific delivery ⇒ decreased efficacy d. The uptake of naked nucleic acids into cells is low ⇒ decreased efficacy 63. What is a lipopeptide nanoparticle? (1 mark) a. A particle composed of synthetic lipopeptides, phospholipids, cholesterol, and pegylated lipids used to deliver nucleic acid therapeutics to cells as a non-viral delivery mechanism. The therapeutic is entrapped within the particle, then delivered to cells. 64. What is bacterial-mediated RNAi? (1 marks) a. A non-viral delivery strategy for nucleic acid therapeutics in which non-pathogenic bacteria are engineered to express shRNA in vivo, to initiate RNAi and decrease expression of a target gene.  65. What is one advantage of bacterial-mediated RNAi for nucleic acid delivery? (1 mark) a. The nucleic acid therapy evades degradation by exogenous nucleases because it is “hidden” within a bacterial cell. 66. What are bacterial minicells? How are they generated? (2 marks) a. Bacterial minicells are small cells that contain no chromosomal DNA, derived from mutant bacteria that use an altered site of cell division. b. When the mutant bacteria is transformed with a nucleic acid, the nucleic acid will be partitioned into minicells. 67. What is a dendrimer? (2 marks) a. A dendrimer is a biocompatible, non-immunogenic nanoparticle with modifiable amine functional groups at its terminal ends. These amine groups act as binding sites for nucleic acids (eg. plasmid DNA, shRNA, siRNA). 68. Describe one example of an antibody-linked nucleic acid delivery system (1 mark). a. Nucleic acids can be linked to antibodies by engineering the antibody to be linked to a positively charged protamine group. The protamine group will allow negatively charged nucleic acids to associate and be carried to the target by the antibody. Unit 4: Vaccines 69. What is a vaccine? How do they work? (2 marks) a. A vaccine is an agent that can be administered to recipients to prevent pathogen infection. b. They work by inducing the recipient to generate antibodies against a disease-causing pathogen. Then, during subsequent exposures the infectious agent is killed by the immunized immune system and the diseased state does not develop. 70. Who are the “fathers of vaccines” and what did they do? (4 marks) a. Edward Jenner → developed a vaccine against smallpox when he realized that milkmaids who had been exposed to cowpox were also immune to smallpox b. Louis Pasteur → developed vaccines against anthrax and rabies 71. List and briefly describe all types of traditional and recombinant vaccines (5 marks). a. Traditional vaccines include inactivated and live-attenuated vaccines. i. Inactivated vaccines immunize recipients with pathogens that have been heated or chemically inactivated. ii. Live-attenuated vaccines immunize recipients with pathogens that have been weakened or made to be less-virulent. b. Recombinant vaccines include subunit, peptide, and genetic vaccines. i. Subunit vaccines immunize recipients with one component of a pathogen, rather than the entire pathogen (eg. a surface protein from a viral particle). ii. Peptide vaccines are a class of subunit vaccines that immunize recipients with a short sequence of 10-20 amino acids, rather than an entire protein subunit.  iii. DNA and RNA vaccines immunize recipients with nucleic acid to be transcribed and/or translated in the cell to produce an antigen 72. How are traditional, live-attenuated vaccines created? (2 marks) a. Traditional live-attenuated vaccines can be prepared by administering the pathogen to a non-human species (eg. monkeys), letting it proliferate and adapt to the new host for several generations, then administering it as a vaccine to humans. b. The idea is that the pathogen will be less pathogenic in humans, but retains its antigenicity to induce an immune response against the original state of the pathogen. 73. Describe four limitations to traditional vaccines (4 marks). a. Not all infectious agents can be grown in culture → makes them difficult to study and design a vaccine against b. Production of animal and human viruses requires animal cell culture ⇒ expensive c. The yield and rate of production from animal cell culture is low ⇒ expensive d. Personnel cannot be exposed to the pathogen ⇒ safety precautions are required e. Insufficient killing or attenuation risks spreading the pathogen f. Even properly attenuated strains can revert to the virulent state, or become cross-contaminated with a different pathogen during preparation ⇒ risks administering a pathogen into recipients g. Require refrigeration and have a limited shelf life 74. What is the main drawback to all recombinant vaccines? (1 mark) a. It takes a long time for new vaccines to reach the market because there are few vaccine manufacturers, the potential for profit is low, and clinical trials are expensive and time consuming. 75. Explain one advantage and one disadvantage to subunit vaccines (2 marks). a. Advantages to subunit vaccines include: i. There is no chance of injecting the viable pathogen and unintentionally infecting them ii. Using a purified protein (free from an extraneous proteins or components) limits undesirable side effects for the recipient b. Disadvantages to subunit vaccines include: i. The purified protein may not have the same conformation in the vaccine as it does as part of the whole pathogen ⇒ may be ineffective at conferring immunity ii. Yield of protein purifications can be low ⇒ makes these vaccines expensive to produce iii. The use of a subunit vaccine applies strong selective pressure in favour of pathogens with mutant versions of that subunit ⇒ these vaccines can quickly become ineffective 76. Describe one example of a subunit vaccine (2 marks). a. The HSV-I subunit vaccine immunizes recipients with a portion of glycoprotein D.  b. The cholera subunit vaccine immunizes recipients with four inactivated V. cholerae strains and a recombinant toxin B subunit. c. The SARS subunit vaccine immunizes recipients with the 192 aa external portion of the spike protein. d. The S. aureus subunit vaccine immunizes recipients with several surface proteins from disease-causing strains. e. The HPV subunit vaccine, Gardasil, immunizes recipients with L1 capsid proteins of four different HPV strains, which self-assemble into virus-like particles. 77. What are three limitations of peptide vaccines? (3 marks) a. 10-20 aa peptides are too short to be recognized by the immune system by themselves → however, this can be solved by linking many copies of the peptide vaccine to a carrier protein (eg. BSA, KLH) or an anti peptide carrier (eg. gold particles) b. To design a peptide vaccine, there must be an epitope recognized by antibodies, that consists of a short consecutive stretch of amino acids → this is often not the case, because the 3D structure of proteins is generated by folding of the primary structure c. A single epitope is not always sufficiently immunogenic to confer immunity to the whole pathogen 78. Describe one example of a peptide vaccine (2 marks). a. The first malaria peptide vaccine immunizes recipients with the most antigenic regions of surface protein 3, to target P. falciparum infection of red blood cells. b. The second malaria peptide vaccine (the RTS,S vaccine, produced by GSK) immunizes recipients with C-terminal amino acids of the RTS,S antigen, fused with the AS01 antigen from Hepatitis B as an adjuvant, to target P. falciparum infection of the liver cells. 79. List four advantages of genetic immunization (ie. DNA/RNA vaccines) (4 marks). a. Enable long-term immunity without the need for frequent boosters b. Does not require cultivation of pathogens ⇒ safer, and possible to design vaccines for pathogens that cannot be cultured c. No chance of injecting the viable pathogen ⇒ safer, especially for young and immunocompromised individuals d. Does not require protein purification ⇒ cheaper vaccine production e. DNA is more stable ⇒ storage is simpler and less expensive f. Several antigens/vaccines can be injected at the same g. The antigen is produced in vivo → for viruses especially, this means post-translational modification occurs exactly as it normally does h. DNA vaccines confer immunity by stimulating antibody production and activating cytotoxic T cells BIOL 432 Test 2 Study Questions Unit 5: Industrial and Environmental Microbiology