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Biotechnology > Midterm Review > Flashcards

Midterm Review Flashcards

1
Q

What are biologics?

A

a substance made from a living organism or its products that is used in the prevention, treatment, or cure of disease of human beings
ex) vaccines

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2
Q

What are small molecules?

A

low molecular weight organic compounds made through highly reproducible processes involving chemical synthesis

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3
Q

What are the advantages and disadvantages of biologics and small molecules?

A

Biologics:
A: interactions with difficult targets like PPIs, specificity for a particular target (small molecules can’t differentiate between related PPIs)
D: not oral, more expensive, high molecular complexity = intricate manufacturing that depends on living organisms

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4
Q

Types of biologics

A
  1. Recombinant proteins replace missing/dysfunctional protein
    - Humalog: modified human insulin
    - Somatotrophin: human growth hormone
    - Factor VIII (hemophilia)
    - EPO (RBCs) for anemia
  2. Monoclonal antibodies block large target molecules
    - Etanercept (RA)
  3. Enzymes
    - pulmozyme: purified DNase used to decrease the viscosity of lung mucosal layer in CF
    - Botulin Toxin
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5
Q

How does Etanercept work?

A

competitive inhibitor of TNF; used to treat RA; produced in CHO cells

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6
Q

How does Botulin Toxin work?

A

has metalloprotease activity that cleaves SNAP-25 (which is part of the SNARE family), resulting in no neurotransmitter (acetylcholine) release, thus paralyzing the muscle

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7
Q

Examples of biologics in industry

A
  1. enzymes like lactase (glucose+galactose), amylase (starch breakdown), cellulase (hydrocarbons into sugars)
  2. biopolymers like xanthan gum
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8
Q

What are biosimilars and what are the challenges?

A

A biosimilar is a biologic that is highly similar to and has no clinically meaningful difference from an existing biologic
(can only be sold after the patent of the original biologic is no longer valid)

advantages
- economic savings in development (more efficient manufacturing process, already FDA approved, low risk)

disadvantages
- process differences may result in unpredicted effects (temp, pH, etc.)
- post-translational modifications could influence binding to target, half-life, aggregate formation, immunogenicity

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9
Q

How is a biosimilar established?

A

comparable quality, PK/PD, efficacy, safety

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10
Q

What is a stem cell?

A

a single cell that can replicate itself or differentiate into many cell types

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11
Q

What are the 3 properties of SCs?

A
  1. capable of dividing and renewing themselves for long periods
  2. unspecialized
  3. give rise to specialized cell types
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12
Q

What is cellular differentiation?

A

Maturation process of primitive cells into the specialized, functional cell types of the body, such as when the blood stem cell produces red blood cells, white blood cells, and platelets

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13
Q

Types of stem cells?

A
  1. totipotent: maximum potential; give rise to any type of cell; fertilized egg and cells up to 8 cell stage
  2. pluripotent: potential to make any differentiated cell in the body (ectoderm, endoderm, mesoderm); ES
  3. multipotent: restricted fate to becoming one of a few types of cells; bone marrow
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14
Q

How do you isolate ESCs?

A

fertilization, remove ICM, grow in dish, change culture conditions to stimulate cells to differentiate

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15
Q

What are iPSCs? How do you make iPSCs?

A

a type of pluripotent cell made directly from a somatic cell

  1. harvest fibroblasts
  2. infect with Oct4, Sox2, Klf4, and c-Myc TFs
  3. correct unwanted mutations by gene targeting
  4. differentiate into embryoid bodies
  5. transplant corrected version back in
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16
Q

How do you make a liver organoid according to the paper we read?

A

iPSCs put under conditions with different factors to produce hepatic endoderm, mesenchymal cells, and endothelial cells that produce liver organoids

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17
Q

What is a Pharmacophore?

A

a part of a molecular structure that is responsible for a particular biological or pharmacological interaction that it undergoes

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18
Q

Why would companies want to make similar drugs?

A

differences in chemical composition of drugs based on same pharmacophore can cause differences in:
1. dosages needed to bring about the drug’s effect
2. uptake by different tissues and cells
3. duration of the drug’s effects in the body
4. side effects and toxicity
5. protocols and costs for synthesis, purification, storage, formulation
6. intellectual property – allowing different companies to sell similar drugs (composition of matter/patent scavenging)

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19
Q

How to discover a new drug?

A
  1. harnessing known or testing novel natural products (Aspirin)
  2. Identify novel druggable and validated targets and employ large-scale chemical screening methods to discover new chemical compounds (NCEs) = Gleevac
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20
Q

What are the problems with natural products?

A

active agent must be identified, purified, and synthesized

target of action might not be known even if physiological effects are

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21
Q

What is pharmacokinetics?

A

what the body does to a drug
- duration, max tolerated concentration, min effective concentration, AUC (drug exposure)
- ADME = (absorption, distribution, metabolism, elimination)

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22
Q

what is pharmacodynamics?

A

the effect of a drug on the body
potency: amount of drug necessary to produce a biological response of a certain magnitude
efficacy: ability of the drug to produce a response by the activation of the receptor

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23
Q

What are the stages of a clinical trial?

A

drug discovery

preclinical: research on a new drug usually done on animals to learn about mechanisms of action, determine how well the treatment works, and see if it is safe to test on humans

Phase 1 (safety): Researchers test an experimental drug or treatment in a small group of people (approximately 20-80) for the first time to evaluate its safety and identify side effects

Phase 2 (efficacy): The experimental drug or treatment is administered to a larger group of people/animals to determine its effectiveness and to further evaluate its safety.

Phase 3 (larger sclae): The experimental drug or treatment is administered to a large group of people/animals (300-3,000 or more) to confirm its effectiveness, monitor side effects, and compare it with standard or equivalent treatments. And compare to certain patient populations

Phase 4: Phase IV: Already FDA approved drugs–look at safety over time and other aspects of the treatment, such as quality of life or cost effectiveness.

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24
Q

what are some characteristics of clinical trials?

A

blind vs. double blind
randomization of participants
comparison to standard of care vs. placebo

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25
Q

Why invest in healthcare?

A

causes of mortality and innovation: infectious disease, cardiovascular disease, cancer

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26
Q

What does the modern biopharma environment look like?

A

originally, big pharma did everything in-house: major R&D, new medicines all the way and commercializing them

now, fund VC startups to do discovery to early clinical trials; “de-risked” enough for large pharma to buy

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27
Q

What is venture capital?

A

a type ofprivate equitycapital provided by outside investors to new businesses that promise to grow fast. Venture capital investments are usually high risk but offer the potential for above-average returns

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28
Q

How do VCs make money?

A

2% management fee: operations, salaries, etc.

20% performance fee aka carried interest: 20% of returns to partners, 80% to LP

look for 3x returns

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29
Q

What is DNA sequencing and synthesis?

A

reading vs. writing DNA; from template without template

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30
Q

Why is sequencing easier than synthesis?

A

NGS is rapid and accurate in determining nucleotide sequences from DNA templates. Can process longer continuous stretches of DNA

DNA synthesis: creation of DNA molecules without a pre-existing template is more complex, has size limitations, increased error rates

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31
Q

What are the two methods of DNA synthesis?

A
  1. Phosphoramidite chemistry: adds one nucleotide at a time to a growing chain using a solid support to immobilize it
    - Deprotection, add nucleotide 3’ to 5’, oxidation, wash excess reagents, repeat
    - Iteration of these steps until the desired DNA sequence is synthesized
  2. Enzymatic DNA synthesis: oligos are systematically synthesized in a cyclic, 2-step process
    - Elongate: terminal deoxynucleotidyl transferase (TdT) adds a single nucleotide to iDNA
    - Deprotect: reversible terminator is removed and ready for elongation
    **faster, greener version
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32
Q

What is the significance of using a solid support in DNA synthesis?

A

allows for the sequential addition of nucleotides, with repeated cycles of washing to remove excess reagents; streamlines the synthesis process

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33
Q

What are the limitations of phosphoramidite chemistry?

A

efficiency drops with length of sequence; large-scale assemblies have to be constructed in stages from smaller strands

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34
Q

What is column-based solid-phase oligonucleotide synthesis?

A

high-throughput, column-based system that allows for the parallel synthesis of multiple oligonucleotides

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35
Q

Steps of column-based solid-phase oligonucelotide synthesis?

A
  1. attachment of an initiator nucleotide to the CPG beads (support) in each column
  2. Successive rounds of nucleotide addition in the form of a phosphoramidite (capping, oxidation, deprotection)
  3. Cleave from column gives stocks of individual oligos
36
Q

What is array synthesis?

A

microarray-based oligonucleotide synthesis
Thousands to millions of DNA sequences are synthesized in a high-density array format on a solid support

37
Q

Compare/contrast the three types of DNA sequencing

A

1) Sanger Sequencing:
- Sequencing by interrupted synthesis using blocked ddNTPs
-DNA synthesis is terminated randomly with each incorporation of a fluorescently labeled ddNTP.
- Process: Utilizes DNA polymerase, primer, and a mix of regular dNTPs and blocked ddNTPs.
- Output: Produces a series of fragments of varying lengths, which are separated by size
- Accuracy: Generally high accuracy but limited read length; cheaper especially if you know what you want to sequence (primer annealing)

2) - Next-Generation Sequencing (NGS):
- Illumina: Sequencing by synthesis = cyclic addition of fluorescently labeled single nucleotides
- Process: DNA fragments with adapters bind to flow cell oligos, form bridge, polymerase creates complementary strand, amplified into clusters, fluorescently tagged nucleotides added, imaged, cleaved, repeated and aligned to reference genome for analysis
- Output: Generates massive amounts of short reads, typically up to a few hundred base pairs in length.
- Accuracy: Generally high accuracy for individual short reads, but errors can accumulate over longer stretches of DNA; expensive; used for whole genome sequencing

3) PacBio and Oxford Nanopore Sequencing:
- Methods: Both are single-molecule sequencing technologies.
- Principle: DNA sequencing by detecting changes in electrical current (Nanopore) or by observing fluorescently labeled nucleotides (PacBio).
- Process: Direct sequencing of DNA without the need for amplification or fragmentation.
- Output: Longer reads compared to traditional NGS, providing more contiguous genome assemblies
- Accuracy: Lower accuracy compared to Sanger sequencing; longer reads more efficiently

38
Q

What is Sanger Sequencing?

A
  1. DNA is denatured into single strands
  2. DNA polymerase extends the primer with standard nucleotides (dNTPs) and labeled ddNTPs.
  3. Termination occurs randomly from ddNTP.
  4. Fragments are separated by size using gel electrophoresis.
  5. The sequence is determined by detecting the fluorescence of labeled ddNTPs as they pass a detector.
39
Q

What is sequencing by synthesis?

A
  1. Template Preparation: Fragmented DNA is linked to adapters, creating sequencing templates.
  2. Clonal Amplification: Templates are amplified on a surface, forming clusters of identical fragments.
  3. Sequencing Cycles:
    a. Nucleotide Addition: Fluorescently labeled nucleotides are added sequentially, with incorporation detected by fluorescence.
    b. Imaging: Clusters are imaged to identify the incorporated nucleotide.
    c. Cleavage: Fluorescent labels are removed to prepare for the next cycle.
  4. Data Analysis: Fluorescence signals are analyzed using bioinformatics to generate DNA sequences.
40
Q

DNA sequencing applications?

A
  1. DNA as probes to measure biological information (MERFISH; RNA in cells + tissues)
  2. DNA as protein detecting aptamers (SOLEX; proteins)
  3. DNA nanotechnology
    - Applications: computation, bioimaging, drug delivery
  4. DNA as a digtal data storage device
  5. DNA-encoded chemical libraries
  6. circulating tumor DNA analysis for non-invasive cancer detection
41
Q

What was the 1000 Genomes Project?

A

create a complete and detailed catalogue of human genetic variations, genotype and phenotype associations, and variance
3 parts
- high coverage sequencing of family trios (heredity)
- high coverage whole exome sequencing (functional consequences)
- low coverage whole genome sequencing (broad overview; common genetic variants)

42
Q

RNA sequencing?

A

oligo primer with T repeats binds to poly-A tail of mature mRNA, reverse transcriptase turns it back into cDNA, amplify and sequence it
**poly-A tail unique to mRNA

43
Q

Different types of RNA-sequencing?

A

Bulk RNA-seq: gives an averaged view of gene expression across all cells in a sample but doesn’t provide information about individual cell types.

Single cell RNA-seq: lets us look at the gene expression of individual cells

Spatial transcriptomics: look at what genes are being expressed and where within the tissue
- maintaining the spatial organization of the cells, which is crucial for understanding the complex architecture and function of tissues

44
Q

How does the data look from the different RNA-seq methods?

A

Bulk: matrix with samples on one axis and gene expression counts on the other

ScRNA-seq: cell type and expression grouped

Spatial transcriptomics: the levels of gene expression and the precise 2D location of these expression levels within the tissue’s architecture (cell + coordinate)

45
Q

How does single-cell RNA -seq work?

A

Cells are suspended with beads containing oligonucleotides and cell labels, single cells and beads form droplets, droplets lyse and mRNA hybridize on beads, reverse transcriptase forms cDNA, sequence

46
Q

How does spatial transcriptomics work?

A

Mount tissue sections onto a solid support, introduce spatially barcoded probes to bind mRNA molecules in the tissue, reverse transcriptase into cDNA, amplify and sequence

47
Q

What is GWAS?

A

associates variations with phenotypic traits; visualized with Manhattan plots
goal = to find genome areas associated with disease

48
Q

What is the missing heritability problem?

A

single genetic variations do not explain the observed heritability of diseases and behaviors

2 possibilities
- genetic susceptibility depends on combinatorial effects of all genes
- genetic contributions to heritability is overestimated

49
Q

How is the missing heritability problem being addressed?

A
  1. develop polygenic risk scores
  2. increase power of studies: sequence full genomes in larger cohorts
  3. measure more modalities: epigenome, repeat regions
50
Q

What is the difference between prognostic markers and predictive markers?

A

what the patient’s outcome is in the absence of therapy vs. if you give this therapy, this person will respond better

many prognostic markers are not predictive markers

51
Q

What is the impact of precision medicine?

A

oncology, molecular measurements to stratify patients, prenatal genetic testing and ethical considerations

52
Q

What are the challenges to making Big Data in biology useful?

A

storage, transfer, access, analysis
want to create an accessible repository of standardized data
- GenBank

53
Q

How does a neural network work?

A

Artificial neurons inspired by biological neurons
Multiple inputs are received from other neurons
The nucleus calculates an activation function
The binary output feeds as input to the next perceptron

limitation: one layer

54
Q

What is the difference between deterministic models and probabilistic models?

A

assume a precise set of reactions and interactions that occur without randomness based on ordinary differential equations; predict an exact outcome given an initial set of conditions, focusing on the average behavior of a large population of molecules; used when a system is well-understood; more rigid

acknowledge the inherent randomness and stochasticity in biological processes, especially at low [reactants] using stochastic differential equations; captures variability and distribution of outcomes; insight into dynamic systems (gene expression, signal transduction)

55
Q

Why do we need predictive algorithms in biology?

A

need ML to create some predictive capability for how a drug will act on the cell

56
Q

Why focus on preclinical models?

A

Only 5-10% of the most promising preclinical studies translate to viable clinical applications
Improving preclinical models to be more physiologically relevant to humans may increase the percentage of studies that make it into clinical practice

want to predict toxicity

57
Q

What are the advantages of cell culture models?

A
  1. easy to manipulate (drugs, genetic modifications, viruses)
  2. advanced techniques to observe them (microscopy)
  3. can generate materials for medicine (grow neurons, vasculature, tissue grafts)
58
Q

How can cells be alive in a dish?

A

actively dividing, metabolically active, can respond to stimuli, have normal structure/order, can grow and differentiate

59
Q

What is viral tropism?

A

what type of cell, tissue, or host it can infect
Pick a cell line that a virus infects to use in culture
ex) polio and HPV can infect HeLa cells, but does not kill them

60
Q

Primary vs. immortalized cells?

A
  1. More natural; from patient tissue; hard to isolate + keep alive (ex vivo)
    Steps: Tissue acquisition, dissection, disaggregation, incubation/growth → not good for long term cells
  2. Basic mechanisms, cancer
    Disadvantages: no healthy immortalized cells, created from tumors so have uncontrollable proliferation + genetic damage, drugs are better at killing healthy cells over cancer cells, so can’t study the effects of cancer drugs preclinically
61
Q

What are organoids?

A

3D cell cultures grown from stem cells or tissue that mimic the structure and function of specific organs or tissues

provide the ability to retain structure of and the different cell types found within the tissue while also providing an easy to manipulate system

62
Q

What are organoids used for?

A
  1. Regenerative medicine
  2. Transplantation
  3. Drug assays (blood-brain barrier and kidneys on a chip)
  4. Disease modeling
  5. Host-microbe interaction
  6. Development (placental organoids)
63
Q

How do you make organoids?

A
  1. cell source: tissue-derived or iPSC-derived
  2. matrix: collagen, mimics ECM for cells to adhere and grow
  3. physical cues: provide ECM support and signaling cues
  4. integrating cues: physiological and structural features of an organ
  5. soluble factors: GFs, small molecules
64
Q

How do you measure successful organoid formation?

A
  1. Tissue scale: patterning, wrinkling, folding, crowding, invaginations
  2. mechanical signature (cell scale): volume, elasticity, cortical tension, cell-cell adhesion, position, shape, size
  3. biochemical signature (intracellular scale): measuring functionality, scRNA-seq
65
Q

Animal models?

A
  • Allows for the most physiologically complete picture
  • Deciding what animal model to use heavily depends on the goal of the project:
    ex) If studying infections, need to consider viral tropism (ferrets + flu)
    ex) If studying physiology/human diseases, need to consider which animal has the closest anatomy to a human (pig heart)
66
Q

What is cryopreservation of the ovary for oncofertility?

A

technique to preserve fragments of ovarian tissue that can be reimplanted to restore fertility from cancer treatment during reproductive years

67
Q

Why edit genomes?

A

functional testing of mutations, treatment of human disease, engineering biological systems

68
Q

What is genome editing?

A

Use chromosomes of living cells as templates in which you develop technologies to edit desired genes/pathways rather than creating from scratch (synthesis)

**want to avoid off-target effects

69
Q

What are the main types of eukaryotic nuclease-mediated gene editing technologies?

A

meganucleases, zinc finger nucleases, TALEN, CRISPR/Cas9

inducing a DSB is universal to all of these

70
Q

What is the difference between NHEJ and HR?

A

broken ends repaired irrespective of homology and results in INDELS = mutagenic vs. repair from template of sister chromatids or homologous chromosomes = natural in meiosis

71
Q

What are the steps of NHEJ?

A
  1. Ku70-Ku80 heterodimer binds to broken DNA + recruits DNA-PKcs
  2. DNA-PKc: kinase that phosphorylates Artemis
  3. Artemis: exo&endonuclease that processes DNA broken ends and prepares them for ligation
  4. Ligase joins broken ends together
72
Q

What are the steps of HR?

A
  1. resection: trimming of broken DNA ends to generate single-stranded DNA (ssDNA) overhangs
  2. Strand Invasion: ssDNA into a homologous DNA sequence on another molecule.
  3. Branch migration and new DNA synthesis
  4. Resolution: Resolution of DNA heteroduplex, often through Holliday junction resolution
73
Q

How to make a mouse model using HR?

A
  1. HR between cloned gene with exon and targeting vector with antibiotic resistance gene + tk you want to insert
  2. Transfect into cells; homology between exon and genome of stem cell
    - If tf is inserted, it wasn’t homologous recombination
  3. Treat with antibiotic and kill cells with TF to see which cells took up the targeting vector; treat with Ganciclovir to kill cells with tf
  4. insert ES cells into different mouse to create a chimeric mouse (carrying cells from 2 mouse strains)
74
Q

What are ZFNs?

A

engineered DNA-binding proteins that consist of a zinc finger domain fused to a nuclease domain (FokI)
- ZF domain designed to recognize specific DNA sequence: each finger binds 3 bp + need 3 to provide binding affinity (typically 3-6)
- FokI induces DSB after dimerization
- DSBs stimulate DNA repair mechanisms like NHEJ or HDR

75
Q

What are TALEs?

A

TAL Effectors: TFs with DNA-binding specificities from central domain of tandem repeats with amino acids in position 12/13 being variable

easier to engineer than ZFNs because of DNA-binding domain

76
Q

Basics of CRISPR?

A
  1. Adaptation:
    - Foreign DNA sequences integrate into a CRISPR locus.
    - Invader sequences are often found near Protospacer Adjacent Motifs (PAMs)
  2. Expression:
    - CRISPR locus is transcribed into precursor RNA.
    - Precursor RNA is processed into mature crRNAs
    - tracrRNA and crRNA are fused together into a single-guide RNA (sgRNA).
  3. Interference:
    - Mature sgRNA directs Cas9 to any genomic locus.
    - Cas9 requires a PAM sequence adjacent to the desired target.
    - Cas9 has two cleavage domains: HNH and RuvC.
    - The Cas9-sgRNA complex induces a double-strand break in the target DNA.
    - DSB triggers NHEJ or HDR
    NHEJ = indels for gene disruption or knockout
    HDR = precise genetic modifications by providing a repair template
77
Q

Mechanism of CRISPR/Cas9 gene editing?

A
  1. tracrRNA (recruits Cas9) and crRNA (binds) are fused together into a sgRNA
  2. Cas9 directed to any genomic locus by a 20-24 bp sgRNA near PAM sequence (nGG); 2 cleavage domains cause DSB
78
Q

Advantages of CRISPR/Cas9 vs. ZFN/TALENs?

A

Disadvantages of CRISPR:
- lack of specificity (off-target mutations)
- mutagenesis via NHEJ
- donor DNA templates required
- immunogenic response

Advantages of CRISPR:
- Single protein required (no protein engineering is needed)
- Targeting depends on bp (easy to design guide RNAs)
- Can simultaneously go after multiple targets with mixed gRNAs

Disadvantages of ZFNs/TALENs:
- Requires protein engineering
- Protein-DNA code is more complex
- Multiplexing is challenging

Advantages of ZFNs/TALENs
- High target specificity
- ZFN derived from eukaryotes (lower immunogenicity)
- Smaller than Cas9

79
Q

Multiplex Automated Genome Engineering (MAGE)

A

Nuclease-free gene editing technique that uses single-stranded oligos annealed by single-stranded annealing proteins (SSAPs).
- Relies on cellular mismatch repair for success.
- Involves introducing oligos targeting genome sites, followed by annealing and incorporation during replication.
- Facilitates precise modifications, including mismatches, insertions, and deletions.
- Enables multiplexed editing without inducing double-strand breaks

80
Q

Key Challenges of Nuclease-Mediated Gene Editing

A
  1. CRISPR/Cas are easy to design but suffer from lack of specificity (off-target mutations)
  2. Mutagenic mutations can be created via NHEJ
  3. Donor DNA templates are required to create a specific edit
  4. Immunogenic responses
81
Q

Limits of Genome Engineering Technology with DSBs

A
  1. mutagenic
  2. cytotoxic when scaled
  3. not suited for combinatorial editing
82
Q

Prime editing? Why is this better than DSB?

A

Components: Cas9 nickase domain (mutated cleavage site/inactivating it), pegRNA: one end is gRNA (crRNA), recruits Cas9 = binding site of interest; other end to strand to be edited with the mutation (reverse transcriptase domain)
Steps: Nicking PAM strand that want to edit, pegRNA binds, RT, 3’ flap with edit, 5’ flap lacks edit + cleaved, DNA repair
- Advantages of PE3: sgRNA nicks unedited strand→ edit on both strands

NHEJ introduces deletions; donor template for HR
Nicking one strand means body will automatically use the native strand as a template

83
Q

Synthetic Biology

A

Engineering Biology: the design & construction of new biological parts, devices, and systems, OR the re-design of existing, natural biological systems for useful purposes

Purpose: break down natural circuits into synthetic circuits, put them in new hosts, create different compounds you want

Less expensive and more efficient

84
Q

Key challenges of gene cluster expression in model hosts

A

Native host challenges
- Difficult or impossible to culture in lab
- BGCs are often silenced in lab conditions

Heterologous host challenges
- Availability of input metabolites
- Improper protein folding
- Toxicity of metabolites can kill cell

85
Q

Proposed solution?

A

Single synthetic genetic element (SGE) that functions across diverse hosts
1. bacteria with native pathway
2. computer-aided design + synthesis of pathway (which host? Which components?)
3. expression of SGE in diverse hosts

86
Q

MAGE for synthetic biology?

A

introduce many mutations into the genome at once to rapidly and precisely engineer intended output

Biotechnology (3 decks)

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