exam 2 study guide Flashcards
peptide therapy
used to replace or mimic the functions of naturally occurring peptides, used for well-being and tissue repair (most efficient is subcutaneous injections)
protein therapy
used to replace protein that is abnormal, reduces impact of disease, enhances protein activity
what do idealized peptide drugs have/are
- high potency and selective
- are an agonist (causes activation of receptor, triggering response)
- stable geometry (rigid 3D structure)
- antagonist/inhibitor (obstruct interactions between a ligand and its receptor)
- replace methionine residues
- reasonable PK+PD (how body on drug and drug on body matches with biological intent, dose regime, safety)
- ability to avoid renal clearance (PEGylation)
- Delivery compatibility and patient adherable administration method
Ideal (usually IV or sub Q)
what are the components of a vector (more detailed in study guide)
- Inducible promoter
- Affinity tag
- Gene of interest
- Antibiotic resistance and origin of replication
what does an inducible promoter do
starts transcription in response to a physical or chemical signal
what does an affinity tag do
adds functionality to the protein to promote purification and typically adds a cleavage site to help with tag removal
what is a gene of interest
DNA sequence that encodes for a desired protein that must be in frame with the promoter and tag; ensured via a multiple cloning site
what does antibiotic resistance and origin of replication allow for
allows for successful host cell selection; only successfully transformed cells will survive antibiotic exposure
what are sources of protein production for therapeutic proteins and their pros/cons (pic in study guide of how to choose source)
- E. coli - lowest cost, rapidest cell growth, potential immunogenicity
- Yeast - rapid cell growth, more expensive, lower expression levels
- Mammalian cells - stable cell lines, slow cell growth, low yield
what are the steps of purification of therapeutic proteins
- Cloning and transforming
Clone a plasmid vector, contain the protein of interest, clones are transformed via insertion into bacteria - Culture and crude protein
Proteins are produced as cells are grown, media is collected for secreted proteins (mammalian cells, yeast), lyse cells for soluble proteins (yeast) - Solubilization
- Strip, wash, elute
Used to further isolate the proteins - Gel electrophoresis
Used to access purity throughout the process
what is a phage
virus that infects a bacteria; allows display of a lot of different proteins or peptides
what is phage display
biological technique to evolve proteins using bacteriophages (enables directed evolution)
what is directed evolution
enabled by phage display -> expedites natural evolution by displaying molecules and selecting the best ones
what is phage display used to study
protein-protein, protein-peptide, and protein-DNA interactions
what are the steps of directed evolution
- Construct phage display library and create coated plate
- Binding
- Washing
- Elution
- Amplification - The eluted phages that contain specificity infect new host cells for amplification
- Repeat - for best binding sequence/selected clone
- Enrichment and Purification
what are (9) delivery methods of peptide and protein therapeutics and pros/cons
Pumps
Pro: Precise delivery control
Con: invasive
Ex: insulin
Liquid-jet
Pro: needle-free
Con: pain and bleeding can occur
Ex: insulin, vaccines
Liposomes
Pro: encapsulate peptide for targeted drug delivery, conjugate w PEG
Con: high cost, leakage
Ex: DOXIL
Ultrasound/electric
Pro: precise targeting, increased efficiency, permeability
Con: only efficient when conjugated w microbubbles limiting load size
Ex: transdermal delivery of insulin
Nasal/pulmonary
Pro: ease of use
Con: can have long-term negative effects
Ex: nasal sprays
Microneedle
Pro: no protein denaturation
Con: loss of dosage accuracy, high cost, difficult admin
Ex: insulin or abaloparatide
Chemical
Pro: uses endocytosis
Con: prone to degradation
Ex: AM-111
Oral
Pro: ease of use
Con: not usually viable as delivery method bc of first pass metabolism
Injection
Pro: increased duration of action, less dosing
Con: risk of infection, ouch
Ex: subcutaneous, intramuscular, intravenous
what are the 2 things antibodies are made of and what are their components
FAB: antigen binding fragment
Heavy and light chains
Contains CDR (complementarity determining region) = provides specificity for antibody
Fc: fragment crystallizable region
Has two heavy chains; binds to proteins, cell receptors
Modulates immune cell interactions
what are the 3 main types of antibody action and what do they do
- Neutralization: antibody prevents the binding of the virus to the target
- Opsonization: decorating the pathogen w antibodies to be consumed by macrophages and neutrophils
- Complement activation: antibodies on the surface can help activate the complement system to kill infected cells
5 approaches for mAb production (explained further in study guide)
- antibody engineering
- phage display
- single B cell
- mouse hybridoma
- transgenic mouse
what are 2 modifications for Abs
CDR grafting: transfer of CDR regions to human scaffold
SDR grafting: reduce immunogenicity of CDR grafted
how are therapeutic mAbs used as targeting molecules
- CDR (complementarity-determining region) binds to the antigen
- Variability gives specificity to targets
- Mimic immune systems attack on cells:
Block cell growth
Trigger cell membrane destruction
Flag cancer cells - Intravenous infusion acts as most common route of admin
pros of therapeutic mAbs as targeting molecules
increased safety, diagnostics allow to differentiate between similar antigens, versatile
cons of of therapeutic mAbs as targeting molecules
immunogenicity from repeated admin, expensive, limited applications
what are AAV vectors
Adeno-Associated Viruses: small non-enveloped virus used for delivering DNA to target cells
- AAV is transformed from a naturally occurring virus into a delivery mechanism for gene therapy
- Viral DNA replaced w DNA of interest
pros of AAV vectors
low toxicity, high rate of infection makes it good for delivery, safe strategy for gene therapy, does not disrupt expression of native genes
cons of AAV vectors
immunogenicity, small packing capacity, expensive
general pathway of AAV vectors (6 steps)
- AAV bound to cell-surface receptors
- Internalization (endocytosis)
- Endosomal escape
- Transport to nucleus
Single-stranded AAV
Self-complementary AAV - Transcription
- Recombination or integration into host genome
what are extracellular barriers to gene delivery (3 + solution)
- Unspecific interactions
Genes can come in contact w proteins for degradation/removal
Genes come in contact w blood cells - Endothelial barriers
Genes need to leave the bloodstream and go through endothelium to enter a certain organ - Inflammatory and immune response
Solution: PEGylation and cholesterol as helper protein
what are intracellular barriers to gene delivery (3 + 2 solutions)
- Cellular uptake
DNA can’t get through cell membrane on its own - needs to use other methods; i.e. AAV vectors, lipid nanoparticles - Endocytosis: DNA can enter cells through endocytosis, but endosomal escape is possible
Solution: lipid nanoparticles (in detail in study guide) - Nuclear delivery: transcription of DNA occurs in the nucleus so DNA must cross the nuclear membrane
Solution: viral vectors - modify them to be drug-delivery vehicles (reduces immunogenicity)
what are toxicity and immune response barriers to gene delivery
Pre-existing anti-viral vector antibodies
Viral proteins and nucleic acids are highly immunogenic
what is CRISPR
CRISPR: gene editing tool that can cut, insert, and delete certain gene segments
Clustered Regularly Interspaced Short Palindromic Repeats
7 parts of CRISPR/Cas
- Target DNA: target sequence to be modified
- Guide RNA: recognizes target sequence
- Cas9 protein: cleaves target DNA to break a double-strand
- PAM sequence: initiates binding to target DNA
- crRNA: specifies target DNA by pairing w target sequence
- tracrRNA: binding scaffold for Cas9
- sgRNA: binding scaffold for Cas9
cons of CRISPR/Cas9
lack of specificity; can induce DNA damage, immunogenicity of Cas9
5 steps of CRISPR
- Complex formation
- Complex binds to DNA
- Guide RNA binds to target DNA
- Cutting of DNA
- DNA repair
genome editing + pros, cons, uses
Precise manipulation of DNA sequences to alter cell fates and expressed traits
Pro: cancer therapeutics, prevent inherent diseases, growth in food production
Con: ethical and safety concerns
Use: treating diseases, replacing damaged/diseased tissues
base editing + pros, cons, uses
Single base-pair change via a nick in the DNA
Pro: precise edits, permanent, can use stop codons to prevent gene transcription and expression
Con: limitations in their targetable sites, bystander mutations
Use: precise editing in non-dividing cells, correction of early stop codons
genome regulation + use of CRISPRi and CRISPRa + pros, cons, uses
Gene repression, gene activation
- CRISPRi blocks RNA polymerase to decrease transcription of target regions
- CRISPRa uses transcriptional machinery to increase transcription of target regions
Pro: versatile
Con: pre-existing antibodies against CRISPR, may select for genome-edited cells w undesired genetic changes
Use: map of molecular interaction, network of biomarkers, medicine and drug design
3 differences between genome editing, base editing, genome regulation
- Genome and base editing are permanent (genome regulation is temporary)
- Genome and base editing changes the gene (genome regulation only affects expression, transient and not heritable changes)
- Genome editing changes entire sequence (base editing only changes one base)
what is RNAi
RNA interference: process where gene expression is inhibited by RNA that shuts down protein translation by binding to mRNA
siRNA and miRNA enter cytoplasm and interact with RISC complex
applications of RNAi
- Target cancerous gene sequences
- Treat bacterial diseases
- Identification of gene function
- Diagnostics
steps of RNAi
- dsRNA is introduced into cytoplasm, processed by Dicer which cleaves the passenger strand leaving the siRNA
- siRNA is recruited by RISC complex, unwinds and incorporates into protein-RNA complex
- RISC and siRNA bind to complementary targeted mRNA
- mRNA is cleaved in a specific site and then degraded in the cell, ultimately disrupting protein synthesis of target gene
what is miRNA (more in study guide)
Endogenous single-stranded RNA
Can bind multiple targets, therapeutic agent, biomarker, drug target, and diagnostic tool
Requires perfect match at positions 2-7
Low specificity and sensitivity
Found in RISC
Only has impact on the gene regulation
Mimics or inhibits
what is siRNA (more in study guide)
Exogenous, double-stranded RNA that cells readily uptake
Requires a perfect match along the entire sequence of interest
High specificity - can’t have multiple targets
Used in DNA methylation, histone modification, gene regulation
Process driven by RISC binding
Destroys mRNA
6 delivery approaches for RNAi
- LNP-formulated siRNAs (via IV)
Avoids first pass metabolism - Small-molecule conjugated siRNA
Easier cellular uptake - SubQ injection
Slower release rate into systemic circulation; can enter lymphatic system - Local administration
Extended release of siRNAs
Retention of RNAi targets within local areas - Exosome delivery
Can increase circulation half-life
Improves cellular uptake - Nucleic acid nanostructures
Designed to react to local biochemical signals to deliver drugs to specific targets
main challenges of delivery approaches for RNAi
delivering to cytoplasm, bioavailability, biodistribution, reducing immunogenicity/toxicity
what is Patisiran
- RNAi therapeutic
- Double-stranded siRNA
- Delivered via IV infusion each 3 weeks
- Long-term dosage; durability of effect to slow disease progression
- Treatment for hATTR
- Greater treatment effect for neuropathy and quality of life outcomes
what is Inotersen
- Therapeutic oligonucleotide
- Single-stranded antisense oligonucleotide
- Delivered via subcutaneous injection weekly
- Lower disease progression and reduce deterioration of quality of life
- Treatment for hATTR
- Lower cost, no drug wastage
4 steps to general immune response to pathogen
- Detect to fight off infection
- Protect body and cells
- Memorize previous foreign infections
- Limit the response after the pathogen has been removed
innate vs adaptive immunity
- Innate: prevents entry of foreign material using physical/chemical barriers
Removes via phagocytosis - Adaptive: prevents disease in the future by remembering what those substances look like and mounting a new immune response
what is humoral and cell-mediated immunity
adaptive immunity’s first lines of defense:
- Humoral: production of antibodies by B lymphocytes
- Cell-mediated: destruction of infected cells by cytotoxic T cells
what are + methods of action of antibodies
activated B cells produce these to counter invading antigens
- Opsonization, neutralization, agglutination, precipitation
passive immunity
antibodies given to a person to prevent or treat disease after exposure to antigen (i.e. given from mom to kid through breast milk)
8 steps of mRNA vaccine mechanism
- Insertion of virus or vector
- mRNA delivery to cytoplasm
- Translation
- Assembly of proteins of interest
- Presentation of antigen by antigen-presenting cell
- B cells activated by the antigen to make antibodies
- Identifying real virus
- Killing infected cells via killer T cells
two types of mRNA vaccines
- Non-replicating: don’t have additional protein sequences to induce self-amplification of inserted mRNA sequence
- Self-amplifying: enhanced antigen expression that mimics a viral infection; results in more potent and longer-lasting antigen-specific effects
component selection for SARS-CoV-2 mRNA LNPs
- Ionizable lipids: attract RNA cargo; improve safety and circulation time
- Helper lipids: modulate fluidity and aids membrane fusion (DSPC in covid shot)
- PEGylated lipids: regulate lipid fusion and increase LNP half-life
- Cholesterol (or its variants): stability enhancers; reduces drug leakage
- mRNA (cargo)
problem + solution + limitations of mRNA LNPs
Problem: mRNA is large and negatively charged: cannot pass through the lipid bilayer of cell membranes; targeted by immune system and faces nuclease degradation
Solution: lipid nanoparticles!
Limitations: transportation, stored in very cold temps, risk of allergic reactions, viral variants, need for boosters
what are microfluidics
enable formulation of small-sized nanoparticles with low dispersity and high encapsulation efficiency; generates reproducible nanoparticle formulations; no other scalable and reliable lab techniques for LNP production
procedure for microfluidics for production of LNPs
- Precipitate lipids in ethanol (ionizable lipid, helper lipid, PEGylated lipid, cholesterol)
- Place mRNA/cargo in aqueous phase
- Mix aqueous and organic phase via fluid flow that meets at a focus
- Ionizable lipid protonated and is attracted to the mRNA which is anionic
- Self-assembly of lipids into nanoparticles due to poor solubility in aqueous to bring along cargo
- LNP are filtered to remove non-aqueous solvent
allogeneic therapies + pros, cons, uses
Derived from same species (i.e. healthy donor)
Pros: fits biopharmaceutical model
Cons: High risk of facing disease, immunological rejection, elimination of donor cells
Used for RBC and platelet therapy
autologous therapies + pros, cons, uses
Derived from patient (personalized)
Pros: personalized, lower risk of immune response, no need for immunosuppressive therapy
Cons: Limited source, cost, requires scale up
Used for regenerative medicine and tissue engineering
CAR-T cell therapy
Chimeric Antigen Receptor proteins engineered to enable T cells to target specific antigens; allows own immune system to fight off cancer
3 components of CAR-T cell therapy
- Antigen recognition region
Extracellular, functions as the receptor, composed of light and heavy chains - Transmembrane region
Anchor for recognition region - Signaling domain
Intracellular, enables tumor cell killing, cytokine production, cell proliferation
6 steps of CAR-T cell therapy
- Remove T cells from patient (autologous)
- Insert gene to express chimeric antigen receptor on surface of T cells
- Validate expression of CAR and select for successful transduction
- Expand CAR T cell population
- Administer CAR T cells to patient via IV
- CAR T cells target cancer cells
6 biomanufacturing challenges
- Long time for manufacturing due to taking blood at one place, processing at another, shipping back and infusing
- Complex: multistep, specialists involved
- Scalability: difficult bc personalized medicine is patient specific
- Cost: $475K for each treatment
- Storage and logistics of keeping cells alive (i.e. keeping them cold)
- Contamination: each sample must be kept on its own and sterile
5 types of clinical trials for stem cell therapies
- Hematopoietic stem cells (HSCs): treatment for blood disorder (44.2%)
Immature cell that can develop into all types of blood cells - Mesenchymal stem cells (MSCs): stem cells found in bone marrow (45.7%)
Multipotent cells - differentiate into many cell types
Can regulate immune responses and have been approved for tissue repair - Limbal stem cells (LSCs): treatment for LSC deficiency
Less prevalent
Multipotent and preserve expressions of genes after cultivation - Neural stem cells (NSCs): used to stave off brain damage and spur functional recovery (1.6%)
Multipotent, self-renewing cells used to generate neurons - Bone marrow derived stem cells (BMCs): multipotent cells isolated from the bone marrow of the donor (2.6%)
Approved for treatment of cancer and immune system disease
