exam 2 prep q's

Question 1

components of a plasmid suitable for therapeutic protein expression

Answer 1

• Inducible promoter
• Affinity tag (+/-)
• Gene of interest
• Antibiotic resistance/other selection mode
• Replication sequence

Question 2

characteristics of a protein that is amenable to rational design modifications

Answer 2

well-characterized sequence and structure (e.g. insulin)

Question 3

describe how directed evolution is used to generate mAb candidates (5 steps)

Answer 3

1. Create a “library” of millions of different phage clones by inserting foreign DNA into the plll
   coat protein (i.e. phage coat protein) gene, the corresponding peptide or protein will be
   displayed
2. Find fusions that bind some immobilized target of interest
3. Wash extensively to get rid of non-specific binders
4. Elute the specific binders and let them infect more bacteria to make more (amplification)
5. Sequence DNA that was in tightest binders to identify sequence to engineer into mAbs

Question 4

components of mAb (sketch in pic on laptop)

Answer 4

antigen binding - Fab, disulfide bridges, CDRs
immune function and lysosome escape - Fc, flexible hinges, glycosylation

Question 5

difference between gene delivery/therapy and genome editing

Answer 5

• Gene delivery – insert new gene into nucleus to enable transcription
• Genome editing – deliver guide RNA, enzyme +/- coding DNA to alter genome (by deletion,
  insertion, or a combo of both)

Question 6

why are both miRNA and siRNA under development as RNAi therapeutics

Answer 6

miRNA less specific but can regulate more pathways, beneficial for multifactorial targets where a single pathway is insufficient; siRNA opposite

Question 7

compare the delivery barriers for RNAi and gene therapy approaches

Answer 7

cytoplasm vs nuclear delivery

Question 8

contrast RNAi and oligonucleotide therapeutic approaches

Answer 8

double vs single-stranded, RNAi can be fully or partially complementary vs ASOs fully complementary

Question 9

mechanism of SARS-CoV-2 vaccine, from delivery to activation of the immune response

Answer 9

• delivery within LNP enables entry to cell and endosomal escape
• mRNA in cytoplasm codes for spike protein expression on cell membrane
• spike protein recognized by circulating lymphocytes

Question 10

components of chimeric antigen receptor + functions of each

Answer 10

• Antigen recognition (scFv targeting CD19)
• Transmembrane domain (CD28; connects antigen recognition with activation)
• Signaling domain (CD28/41BB initiates T cell activation/CD3z enables tumor cell killing; also
  enables cell survival and proliferation, cytokine production)

Question 11

benefits of allogeneic cell therapies compared to autologous

Answer 11

cheaper, off-the-shelf, more “drug-like”

Question 12

what are some barriers to extracellular vesicle therapeutic development

Answer 12

• Lack of mechanistic understanding
• Scale-up biomanufacturing
• Purification quality control