Therapeutic Molecules and Antibodies

Native Function of CRISPR/Cas9 System

• CRISPR naturally acts as an immune system in bacteria.
• It involves a CRISPR array and CRISPR-associated proteins like Cas9.
• The CRISPR array has variable spacer regions separated by repeat regions; the variable spacer regions come from past bacteriophage infections.
• During a bacteriophage infection, the CRISPR array is transcribed, creating crRNA fragments.
• If the bacteriophage has infected the bacterial lineage before, one of the crRNAs will be complementary to the bacteriophage genome.
• The crRNA will associate with Cas9 nuclease, guiding it to the matching spot on the bacteriophage genome.
• Cas9 will then cleave the bacteriophage genome near where the crRNA binds, stopping the infection.
• If the bacteriophage is new to the bacterial lineage, Cas9 will cleave at PAM sites without crRNA binding. This generates a fragment of the genome that can be added into the CRISPR array for future defense.

Biotechnological Use of the CRISPR/Cas9 System

• The Cas9 enzyme and a guide RNA (gRNA) specific to the target site can be introduced.
• This can be done by transforming or transfecting the target organism/tissue with an expression vector carrying the Cas9 and gRNA genes.
• Once inside, the gRNA associates with the Cas9 nuclease, which scans the genome for PAM sites.
• If the gRNA is complementary at a PAM site, Cas9 introduces nicks or cuts, resulting in a double-stranded break upstream of the PAM site.
• The cell tries to repair the dsDNA break, using either homology directed repair (HDR) or non-homologous end joining (NHEJ).
• In NHEJ, the cell joins non-homologous DNA ends; insertions and deletions can occur, knocking out the gene where the break occurred.
• In HDR, a homologous sequence repairs the break; donor DNA containing regions homologous to the flanking regions is provided. Donor DNA can carry a mutation or a gene to be introduced.

PAM Site Function and Location

• A PAM site is an NGG motif that Cas9 nuclease binds to initially when cleaving DNA.
• It is found on the strand opposite the one that the guide RNA binds to.

Guide RNA Components in Native CRISPR

• Guide RNA is made of crispr RNA (crRNA) and tracrRNA (trRNA).
• crRNA is complementary to the target DNA and provides specificity to Cas9 cleavage.
• trRNA anneals to crRNA and binds to Cas9, attaching the guide RNA to the Cas9 enzyme.

Drawback to CRISPR

• Off-targeting is the major drawback.
• Off-targeting is when Cas9 cleaves the genome in an unintended location.

Multiplexing

• Multiplexing is introducing multiple gRNAs during a CRISPR experiment.
• This allows for multiple mutations in one CRISPR iteration, like deleting one gene and introducing a point mutation on another chromosome.

Gene-Editing Techniques besides CRISPR

Zinc Finger Nucleases (ZFNs)

• ZFNs use an enzyme featuring zinc-finger motifs that are specific to the target DNA sequence linked to a Fokl nuclease.
• It requires designing two enzymes for the same location on complementary DNA strands.
• When the enzymes bind, Fokl nucleases are in proximity and cause a dsDNA break.

TAL Effector Nucleases (TALENs)

• TALENs gene editing uses TALE enzymes that are designed to bind to target regions of DNA linked to a Fokl nuclease
• It also requires designing two TALENs for each DNA strand so that the Fokl nucleases are close to one another to cause a dsDNA break.

CRISPR Advantages Over ZFNs/TALENs

• CRISPR only needs selection of a gRNA specific to the target location.
• ZFNs and TALENs both need the design and synthesis of an entire enzyme specific for the target location.
• CRISPR is simpler, quicker, and cheaper.

Components for Introducing a Point Mutation with CRISPR

• Cas9 nuclease carries out the DNA cleavage for repair.
• Guide RNA guides Cas9 to the target location.
• A PAM site is a binding site for Cas9 in DNA close to the gRNA binding site.
• Donor DNA has homologous regions and contains the point mutation to incorporate.

Modified Versions of Cas9

dCas9

• It has both nuclease domains deactivated, so it doesn't cleave DNA.
• Uses include down-regulating gene expression, or up-regulating gene expression, visualizing DNA.
• dCas9 can also be used to introduce site-specific mutations, or induce a dsDNA break.

Cas9 Nickase

• It has one nuclease domain deactivated, so it introduces nicks (ssDNA breaks)
• A pair of these nickases can be used together to target opposite strands in close proximity, behaving like a dsDNA break.
• Off-target mutations are reduced, because the Cas9 nickases must cleave in close proximity for a break to occur

Altering gRNA to Increase Specificity

• Altering the gRNA to be 20 nt long and include a GG motif on the 5' end can increase CRISPR cleavage specificity.
• Reducing gRNA length to 17 nt also improves specificity.

CRISPR 2.0

• CRISPR 2.0 uses Cpf1 as the nuclease.
• Cpf1 introduces staggered cuts similar to restriction enzyme digestion and is more specific than Cas9.

Biohackers

• Biohackers are people who take advantage of biotechnology in their personal lives outside of organized research.

Gibson Assembly

• Gibson assembly is a technique for scarless assembly of multiple DNA fragments in a single reaction tube.
• Fragments are amplified with PCR primers that add homologous sequences to each end.
• Fragments are mixed with Gibson assembly master mix that contains 5’ exonuclease, DNA polymerase, and DNA ligase.
• The exonuclease chews back the 5’ strand generating 3’ overhangs. Complementary overhangs anneal, DNA polymerase fills the gaps, and DNA ligase seals nicks.

Protein Therapeutic

• It’s a protein administered to treat a diseased state.
• Examples of protein therapeutics include Herceptin, Humira, and Rituxan.

Largest Category of Protein Therapeutics

• Antibodies

Drug Development Steps

• The discovery phase learns about the drugs before they are used on humans.
• The initial screening of millions of compounds for a target compound often occurs during the discover phase.
• Then, the preclinical pharmacology phase focuses on research, mode of action, chemical structure, and biochemical properties.
• Following that, the preclinical safety phase is focused on assessing the harm done to small animals, like mice.
• Then, the clinical pharmacology and safety phase involves assessing the harm of different doses done to higher animals.
• The exploratory development and full development phases consist of phase I, II, and III clinical trials on humans.
• Phase I clinical trials tests the drug's safety and finds the dosage that can be given to healthy individuals without serious side effects.
• Phase II clinical trials test proper dosage using test and control groups.
• Phase III clinical trials assess the magnitude of the drugs affect on the disease phenotype.
• After clinical trials, commercialization begins, and the product is able to be sold.

Recombinant DNA Technology in Protein Therapeutics

• Recombinant DNA technology can produce protein therapeutics through modifying expression to produce proteins.
• The advantages are that therapeutics can be manufactured cheaply in large quantities and easily modified. The process is also highly reproducible.

Recombinant Technology in Interferon Therapeutics

• Interferon is naturally made by animal cells in response to viral infections.
• When a viral infection occurs in one cell, interferon is made and secreted. It then interacts with receptors on neighboring cells and induces antiviral protein production.
• Recombinant technology creates hybrid interferon from multiple natural interferons.
• This can be done by using restriction ezymes to re-ligate fragments of several interferon genes together.
• Alternatively, PCR synthesis is used to assembles fragments in a specific order.

Drug Half-Life

• The half-life of a drug is the time it takes for half of the starting dose to be removed from the body.
• Half-life of therapeutics can be increased via pegylation (attaching polyethylene glycol (PEG)) to decrease renal clearance.
• Dimerization can be used to decrease the rate of degradation, increasing half life.
• Half-life of proteins can also be increased by fusing proteins with an XTEN domain. XTEN domains increase stability, solubility, and resistance to aggregation.

Benefits of Increased Therapeutic Half-Life

• An increased half-life means it persists longer in the bloodstream.
• This means dosing less frequently, which leads to reduced costs.

Non-Antibody Protein Therapeutic Examples

DNase and Alginate Lyase

• Used to treat cystic fibrosis.
• Modified DNAsel degrades DNA to thin mucus.
• Modified alginate lyase degrades alginate produced, reducing biofilm formation.

Bacteriophages

• Used to treat cystic fibrosis.
• Delivering a non-lytic bacteriophage carrying an adjuvant gene to make cells antibiotic-sensitive.

IL-10

• Used to treat Crohn’s disease and ulcerative colitis by modulating T-cells.
• LAB is lacking an essential gene so the cell cannot proliferate outside of the human body.

Leptin

• Used to treat obesity in mice by telling the body when there is enough food.

Cyanovirin N

• Used to prevent transmission of AIDS.
• Cyanovirin N is a protein that binds gp120 and prevents entry into host cells.

Bioidentical Insulin

• Engineer LAB to express bioidentical insulin to administer to patients with diabetes.

Immunogenic Recognitions of Foreign Entities

• A macrophage takes in proteins from the extracellular environment.
• Proteins are processed in the endosome to make small protein epitopes.
• Protein fragments bind MHCs and are transported to the cell membrane.
• The MHC complex shows the epitope for recognition by T-cells.
• A T-cell then undergoes a change and initiates a gene expression.

Hybridoma

• It’s a fusion between a plasma cell and a myeloma cell and it can produce antibodies.

Monoclonal Antibody Production

• Inoculate a mouse, rat or rabbit with a wished antigen.
• Euthanize animal, harvest the spleen and isolate B-cells.
• Combine B-cells, myeloma cells and polyethylene glycol to facilitate fusion. Plate the mixture on HAT media which only allows hybridomas to grow. Screen hybridomas and dilute into culture plates by testing supernatant via ELISA.

Hybridoma Selection

• Mix B-cells with myeloma cells and PEG to facilitate membrane fusion, resulting in a mix of various cells.
• Plate on HAT media, which contains aminopterin which blocks enzymes that make dGTP and dTTP. This also contains thymidine and hypoxanthine, which allow the salvage pathway to complete.
• B-Cells are HGPRT+ and can use the salvage pathway and proliferate, however they are not immortal and die quickly.
• Myeloma cells use HGPRT and will not be able to synthesize dGTP or dTTP, and die.
• Hybridomas are HGPRT+ and immortal, so they will be able to synthesize dGTP and dTTP and divide for a long time.

Hybridoma Advantages

• Hybridomas are immortal and do not require regeneration of a monoclonal antibody for the antigen.
• They are a long-term supply of monoclonal antibodies because the cells can be cryopreserved and thawed when required.

Drawbacks of Non-Human Antibodies

• Antibodies made in other organisms have a species-specific Fc region that is not present in humans.
• Generates an immune response that destroys the antibody.

Humanized Antibodies

• Chimeric antibodies fused to the antigen-specific Fv region.
• Humanized are 955% of the time humanized.

Complementarity Determining Regions (CDRs)

• They are hypervariable domains within the Fv region of an antibody.
• Determines the epitope specificity.

Creating 95% Humanized Antibodies

• Use Gibson assembly or PCR synthesis to assemble CDRs into the Fv region of the human antibody, as a result generating 95% humanized antibody.

XenoMice

• XenoMice are humanized mice that have been engineered to produce human antibodies.
• They are a solution to creating human antibodies that aren't recognized by immune-system.

Generating Xeno Mice

• Delete the IgH and IgK genes from mouse embryonic stem cells in order to make an deficient mouse line.
• Then into another mouse introduce human IgH and IgK genes.
• Breed the mice lines and chose offspring that have mouse IgH and IgK deficiencies.
• The result would then produce human IgH and IgK.

Antibody Fragments

• These antibody fragments are Fv and Fc region combinations.
• Smaller fragments mean they are cheaper.
• Easier for tumors to infiltrate.

Diabody

• It’s an antibody fragment composed of two Fv domains but doesn’t use Fab and Fc regions.
• Bivalent diabodies can be composed of two identical Fv domains whereas bispecific diabodies use two different Fv domains.

Antibodies as a Protein Therapeutic

• One drawback to antibodies are that they are therapeutic proteins and proteins degrade quickly.
• An antibody half-life is around 9 days.

Increasing Antibody Half-Life

• Mutagenesis can be carried on a Fc domain to increase half-life.

Antibody Protein Therapeutics

C. diphtheriae

• Antibodies against it are used to treat diphtheriae.

Rituximab

• It’s against the CD20 antigen on the surface of B cells used to treat lymphoma.
• Kills B-cells by binding to them.
• Induces apoptosis,
• Recruits macrophages and killer cells.

Herceptin

• It’s a monoclonal antibody that inhibits the binding of growth factors against the HER2 receptor.
• Prevents uncontroled growth cells.

Select CDRs

• They efficiently target the Epstein-Barr virus.

Antibodies (IL-12)

• Contains two FV regions that are specific to IL-12 and IL-18.

Irinotecan

• It can be administered to induce expression of unique cell surface proteins on colorectal cancer cells.
• Also reduces tumor size.

ROR1 Receptors

• Receptors that are in use that can be found in cancer.
• Only a problem if dimerization occurs.

Abzyme

• The antibodies used target and degrade ghrelin.
• Decreases hunger levels.
• Helps with weight loss.

Abzymes

• Known as monoclonal antibodies.
• They use active sites that possess activity.

Types of Nucleic Acid Therapeutics

Antisense Oligonucleotides

• Lengths of those are between 15-24 nt
• Can target mRNA
• Inhibits translation of the mRNA

Aptamers

• These bind to different motifs on proteins.
• Binds to peptides or ligands that also can bind.

Ribozymes and Deoxyribozymes

• Both can cleave molecules acid molecules.

RNAi

• Directs small RNAs the degradation of mRNAs through sequence-specific degradation.

Characteristics of Antisense Oligonucleotides

• Must be readily taken up by cells.
• Specific to mRNA
• Must be degradation resistance.

Types of Second & Third generation antisense oligonucleotides

• Second generation antisense oligonucleotides
• Have a 2' methoxyethyl group on the 5' and 3' end nucleotide
• Third generation antisense oligonucleotides
• Less toxic, longest half-lives than the last generation.

Guidelines for Anti-Sense Oligonucleotides

• Effective if directed to mRNA's 5' and 3' ends.
• Works well on intron-exon boundaries.
• Optimal effect on naturally double stranded regions.

Nucleic Acid Thereapy

• Antisense oligonucleotides can down regulate target genes
• Bind complementary mRNA is not affected that inhibits the translation
• May be transformed or transfected directly into cells
• Can happen again through vectors.

Advantage Over Antibodies

• These are generally not destroyed by the immune system
• Are based on nucleic-acid.

SELEX Process

• Method for generating.
• Helps for identifying what binds the target molecules.
• Starts by cloning DNA into vectors.
• T7 RNA Transcribes the sequencess into RNA.
• Incubation period happens. And then wash away.
• Amplfiy through PCR, and repeat to enrich it.

Aptamers

• Can modify this area to increase stability.
• Decrease sensitivity to nuclease degradation through 2' methoxy groups through purine capping

Limitations of Aptamers

• One limitation is maintaining levels in-vivo.
• Can improve availablity through pegylation and liposomes.
• In turn that reduces half-life.

Nucleic Acid Thereapy Example

Pegaptanib

• Binds to vascular growth factor for slowing macular degeneration.
• Errors that are usually processed cause vision loss.

Apotamers

• Designed to distinguish between wildtype P52 or Mutant R175H P52 using methods.
• In each iteration that is preffered is bound bound while others were removed.

Lamin A/C

• Binds to siRNA to release that to prostate cancer.
• Degrades cancerous lamin.

Ribosymes & Deoxiribosymes

• Are 40-50 Nt RNAs.
• They both have a catlytic domain.
• Both also are used with deoxyribozymes.

Advantages over Ribozymes

• Deooxy are more efficient at binding rna molecules that are similar.
• Eaiser for synthesizing.
• They also have an easier and higher production.
• They also turn over catalytic

Therapeutic

• Is problematic when delivering Deoxyribozymes and Ribosyzemes to cells that is problematic.

Types of RNAi

Cleavage

• Inhibit expression with cleavage that is known as viral RNA.

DNAzyme

• Can treat cancer.
• Cleaves c-jun and down-regulates the function of the component.

RNA Interference & Degradation of Specific mRNA

• Binds of of a different small mRNA and binds to reduce gene expression

dsRNA

• Introduced in the area the the fragments are known as siRNA through dicer.

Antisense

• Risc complex is guided through siRNA.
• mRNA has an 90% reduction in gene expression.

siRNA

• Used in RNAi vivo
• has one bulged mismatch to prevent interferon causing unpleasant side effects
• should try to stray from high toxicity UGC like sequesnces
• 27/29 are better than 21

Huntington’s Disease

• siRNA’s and mRNAs have an extended CAG that is meant to treat against prevention and aggregation.
• onpattro is an example for hAttrp

Name/Describe Viral Vectors

Gamma RetroVirus

Can be used to intergrade stably within the genome. EX Meleny murinie virus.

Lentivirus

Can be used to intergrade stably within the genome. Specfically targets DIVIDING CELLS EX: HIV-1

Adrenovirus

Infects a variety of Non-dividing cells Can’t intergrade into the host genome.

Adeno-Assocciated Virus

• Non-Pathogeic
• needs coinfection with a virus to operate Intergrade into the genome.

HSV-1

• Does not intergrade into the chromosome/genome.
• Targets neurones specifically.

Events for the Infections in Cells

• The fibers bind to proteins surface
• Vesicles merge through microtubules
• At the pole it is released
• Virus Replication happens
• The the virus is present new viruses form.

What Vector

• HSV-1 because it doesn't infect the genes
• Is capable for tagetting the Neurones

Advantage For Vectors

HSV-1

• Has a high sequesnce and multiple gene copies.
• Dose not intergrade

Non Vrial Delivery

Injection

Lipid

Non Pathogenic

Bacterial

Dendimers

Via Antibodies

Vivia Apotamers

Disadvantage for Injection for HIV

Disadvantage HIV

• Cleared Quickly
• Degradaed by enzymes in serum
• Doesn't have the same effect

Lipopeptide Nanoparticle

• Delivers lipid protein to the cell
• Gets delivered to all cells

Via Bacterial

A non virus that introduces RNA to cause degradation.

Advantage for Viral RNai

Evades Degradation and is Hidden in the bacterial cell.

Bacterial Minicells

• Used from mutant bacterial cells and used with alternative division.
• Nucleic acids transferr and is partitioned.

Dendrimer

• The modifiable amine uses groups bind sites

AntiBodies

• Are linked to by engineering the antibody

Vaccines

• Agents that cause immune system response
• This is caused by the recipients generating the anitbodies.

Who invented

• Edward
• Louis that used anthrax and rabies.

Traditional

Uses pathogens that have been killed by the immune system.

Recombinate

Has sununir Uses the the the and proteins.

Long does vaccines require

This takes a lot of time to manufacture for them and the testing of them.

Explore various therapeutic molecules like ribozymes and antibodies used in targeted therapies. Learn about their mechanisms in treating diseases such as ovarian cancer and HSV. Discover how XenoMice are created for antibody research.