Midterm 1 Flashcards
lectures 1-7
gibson assembly of fragments
- take 2 blunt ends –> PCR amplify using primers to create overlapping regions
- amplicons have overlap –> use matermix enzymes to trim down overlaps –> overhangs
- annealing of overhangs
- DNA pol + ligase to fill gaps + nicks
CRISPR acronym
clustered regularly interspaced short palindromic repeats
crispr gene editting requirements
target sequence
chimeric gRNA (simplification fulfilling crRNA and trRNA fxns)
CAS9 endonuclease (induce DSB)
ssDNA (specific desired mutation flanked by homologous regions –> allow HDR)
PAM site (distinguishes target seq vs bacterial crispr array)
HDR vs NHEJ
NHEJ = randomized indels
HDR = controlled, specified, indels
talens/Zn fingers
peptides that bind to target
DNA binding domains flank site of mutagenesis
linked to FOK1 nucleases –> cover site of mutagenesis
rec domains MUST perfectly overlap –> +/- 1 nt =/= DSB
CRISPRi - gene silencing
inactivated CAS9 –> just sits on the GOI –> blocks RNA Pol –> gene is silenced
remove Cas9 –> gene is reactivated
CRISPR CF1 modification
- use 2 gRNAs –> induce ssDNA nicks in CLOSE proxy –> allows HDR indels
- nicks far apart –> no HDR indel –> independent nicks easily repaired w/o issues –> decreases crispr off-targeting errors
- chances of nicks in close proxy = low –> increased crispr specificity
Interferon for Protein Therapeutics
- Interferons (IFN gene) –> expressed cytokines to alert immune response –> increased efficiency of treatments
- Effectiveness can be further improved using gene shuffled IFN variants –> maximize interferon effectiveness –> combine IFN alpha 1 and alpha 2
- issue with interferons: hard to purify
half life of protein therapeutics
- time for [proteins] to halve
- can be improved using polyethylene glycol (PEG) –> pegylation
- PEG improves protein stability + decreases excretion from body (persists in body longer)
examples of extending half life of protein therapies (alternatives to PEG)
HGH, XTEN protein tag
Extending half life - HGH
modify HGH w/receptor peptide –> causes HGH dimerization
dimerized –> harder to degrade –> increased half life
Extending half life - XTEN tag
Alternatives to Pegylation
protein tag increases stability –> higher half life
allows purification of product using CBD
tag is cleaved off of product to obtain fxnal form
Combating cystic fibrosis
- caused by P. aeruginosa –> forms alginate biofilm –> difficult to deliver drugs
- Soln 1: introduce alginate lyase produced/harvested from Ecoli to degrade biofilm. Bacterially derived enzyme –> immunogenic. Mitigate by mutating epitopes
- Soln 2: use non lytic phage M13 carrying LexA gene. non lytic –> minimal pressure against the phage. LexA interrupts SOS DNA repair –> Become susceptible to quinalone treatments
Crohn’s/Ulcerative colitis treatment
- Delivery of drugs via LABs native to gut microbiome
- integrate interleukin 10 (IL-10) to become thymidylate synthase (-) –> can’t synthesize thymidine –> reliant on patient to scavenge
- integration of IL-10 ensures modified bacteria cannot persist in nature
AIDs treatment
- Cyanovirin N expression using L. bacillis (native to vagina)
- constitutive expression of Cyanovirin N –> binds AIDs gp120 and gp41 receptors –> blocks infection
- improve cyanovirin N protein stability by replacing Gly w/ Pro
Antibody stuff definitions
antigen (Ag) = foreign object
antibody (Ab) = proteins from plasma cells to facilitate epitome-macrophage interactions
epitope = specific structure on the Ag recognized by Ab
antibody structures
heavy chain + light chain
FC (constant, crystalized) region = leukocyte intrx domain
Fab = epitope recognition region
FV (variable) = specific portion of Fab for epitope recognition
harvesting mAb
- inject Ag to mouse –> pAb excreted
- 2 injections to ensure high Ab specificity
- harvest plasma cells from spleen –> fusion by PEG w/HGPRT(-) myeloma
- selection on HAT media for immortalized B cell hybridoma
- dilute to isolate 1 cell/drop –> selection for mAb to target epitope using ELISA
- culture and express mAb of interest
Ab humanization
purpose of humanization = decrease risk of immunogenic response from Ab treatments
- use Fab with human Fab constant region + mouse FC –> partial humanization
- engineer human Ab with FC containing mouse CDR regions –> 95% humanized
OR make humanized mouse
- lineage 1 = delete both IgH and IgK heavy/light chain genes
- lineag2 2 = introduce human IgH and Igk
- crossbreed –> select offspring mice carrying only humanized IgH and Igk –> xenomouse –> produces only human antibodies
optimizing Ab
creation of partial Ab’s
- FC = actual epitope recognition –> dont need the Fab constant domain –> isolate FC
- link 2 using linkage peptide FC –> diabody
- 2 of same FC –> bivalent diabody
- 2 of different FC –> bispecific diabody
example usage of diabodies
- use Irinonectan on cancer cells –> induce cancer-specific cell receptors
- use bivalent diabody. FC1 binds receptor, FC2 binds/carries a toxin –> targetted delivery of toxin to cancer cells
Abzymes
Ab with catalytic FC –> abzyme
example abzyme usage
more ghrelin protein = more hunger
- inject transition state analogue to ghrelin –> mouse excretes enzymatic Ab –> able to cleave ghrelin precursor
- therefore prevent hunger accumulation
antisense RNA
- antisense cDNA –> antisense RNA –> bind transcript –> block expression
- RNA oligonucleotide –> bind transcript –> block expression
improving antisense oligonucleotides
- Replace P with S in phosphatediester bonds –> resistant to nuclease degradation
- capping with methoxyethyl –> resist nucleases + higher target affinity
- introduce deoxynucleotides to form RNA-DNA duplex –> activates RNAseH –> Cleave target transcript
Retroviral life cycle
- retroviral RNA –> reverse transcriptase to form RNA-DNA duplex
- RNA strand degraded –> cDNA used to form retroviral dsDNA
- retroviral DNA integrated into bacterial genome for replication
Interrupting retroviral life cycle using DNA therapeutics
Introduce oligonucleotide to occupy retroviral tRNA site –> block synthesis of cDNA. Therefore no retroviral DNA is synthesized
RNA Aptamers
- Similar to Ab’s –> high affinity for target molecule (protein or other NAs)
- unlike AB’s –> NOT immunogenic (body doesnt react to NAs, only proteins)
creation of aptamers
SELEX selection
- random nucleotide sequences –> express random RNA aptamers
- test on epitope of interest –> find aptamer that successfully binds
- dissociate bound aptamer –> cDNA –> find sequence of aptamer
- PCR the apamter cDNA –> rescreen on epitope –> repeat selection to increase affinity
Ribozymes
Recall: RNA can be enzymatic
- recognition domain to recognize with substrate
- enzymatic domain
RNAi - RISC complex
- introduce dsRNA (or vector expresses hairpin RNA shRNA)
- DICER enzyme cleaves dsRNA –> siRNA
- Argonaute enzyme binds siRNA –> remove passenger RNA –> ssRNA
- Argonaute + recognition ssRNA = RISC complex
- RISC binds target transcript using ssRNA hybridization –> cleave transcript
results in ~90% reduction to expression
Viral methods of NA delivery
RHAL
- retrovirus - encode GOI in retroviral genome –> transform –> GOI as cDNA –> integrate
- lentivirus - removal of nonpathogenic genes –> use to infect non-dividing cells
- adeno-associated virus - TINY virus, relies on helper adeno virus to replicate. Deliver alongside adeno –> replication. No adeno –> integration of GOI
- HSV1 - can carry up to 150 kb of nt –> MASSIVE gene delivery
Viral delivery of NAs - SCIDS
- rare disease - T cell deficiency
- originally treat by using pegylated adeno deaminase (ADA) - ADA stimulates T cells
- PEG causes immunogenic response –> ADA treatment only works short term
- deliver wt IGL2 gene (to repalce mutated ILG2) using retrovirus –> hematopoetic stem cells –> transplant
- retrovirus modified to prevent leukemia side effect
NON viral methods of GOI delivery - non carrier molecules
LMB-Z
- modified zygote carrying GOI –> transplant to surrogate
- liposome nanoparticle - modified with lipoproteins to carry NAs inside
- Minicells - transform with GOI into mutated bacteria–> replicates as minicells –> act as naturally derived liposomes (non immunogenic)
- non pathogenic bacteria - GOI in plasmid + invasin –> invasin gene allows interaction with mammalian B1-integrin receptors
carrier molecules for delivery of GOI
DAAC
- Aptamer
- single chain antibody - FV to recognize target host receptor. FC linked with (+) protoamine (attracts GOI)
- Dendrimer - large non immunogenic molecule (+) charged (carries the GOI) –> endocytosis
- siRNA (for RISC) linked to cholesterol
siRNA - cholesterol delivery - ApoB gene
targetted delivery of siRNA using cholesterol carrier to target ApoB gene –> 50-70% reduction of serum cholesterol
traditional vaccines
heat killed/inactivated virus –> decrease virulence but maintain immunogenicity
attenuated virus –> pass virus through animal –> mutation reduces virulence to humans
limitations: expensive + low product yields, hazardous to culture, not all virusses can be easily cultured, attenuated virus can revert back to regain human virulence
subunit vaccine
overexpression of a speciifc peptide (subunit) epitope to illicit immunigenic response
HIGHLY SPECIFIC to that particular epitope
peptide vaccine
several subunits/fragments linked to a carrier protein (BSA or KLH) –> subunits alone = too small to be recognized by immune system
can use multiple diff subunits to create vaccine for multiple antigenic sites
DNA vector vaccine
encode viral DNA or cDNA in plasmid + CpG adjuvant gene (adj stimulates immune response) + polyG tail to increase uptake of vaccine
plasmid delivered to host –> expression of epitopes/antigenic fragments –> present on MHCI –> T cells respond –> formation of memory cells + antibodies
MIDGE vector vaccine
DNA vector vaccine is cloned using antibiotic selectable marker –> not ideal for human usage –> needs to be removed
excise GOI and cap ends to prevent degradation –> MIDGE vector
