Advanced Technologies Notes PDF

Advanced technologies. Class 1. Unexplored therapeutic opportunities: targeting receptors, transporters, channels and enzymes and etc for systems such as respiratory system, cardiovascular and genitourinary systems. Rituximab (anti-CD20):  monoclonal antibody binding to CD20+ B cells, leading to...

Advanced technologies. Class 1. Unexplored therapeutic opportunities: targeting receptors, transporters, channels and enzymes and etc for systems such as respiratory system, cardiovascular and genitourinary systems. Rituximab (anti-CD20):  monoclonal antibody binding to CD20+ B cells, leading to their destruction by effector functions through various immune mechanisms, including complement(C3b) binding to Rituximab and NK cells can bind to the C3b to induce cytotoxicity and phagocytosis by macrophages that can bind to the Fc region.\>50% Success rate needs to be administered weekly as it is not a cure. monoclonal therapies: such as Rituximab and for checkpoint inhibitors are a very strong market. JAK (tyrosine kinase) Inhibitors: JAK is an enzyme that is used to send signal from receptors such as IL-2R or IL-6R. Several drugs (like Tofacitinib and Ruxolitinib) inhibit these JAK enzymes to reduce inflammation by preventing phosphorylation of STAT for gene expression. Gene therapies pricing and reimbursement : Value-based pricing is centered on clinical trial data and theoretical benefits, while outcome-based pricing relies on real-world effectiveness and patient outcomes. SCID, or Severe Combined Immunodeficiency lack functional T cells and B cells absence of receptors such as IL-2, IL-4, IL-21 due to mutated common gamma chain of Type 1 receptors. SCID-X1: defective gamma chain, this causes defect in the JAK 3 signalling and seen in T, NK cells. Nude: hairless athymic nude mice lack a functional thymus thus lack T cells. How to treat SCID? Autologous or allogeneic bone marrow transplants from immune-competent HLA-matched donors: patient (autologous) or a healthy donor (allogeneic) blood or bone marrow is taken. Stem cells are taken out and incubated with antibodies, stems cells that can bind to the antibody is selected (cryopreserved then thawed for use) diluted and infused into the patient. The autologous cells go through modifying of the HSC by transducing vectors encoding the corrected gene. Conditioning correct B cells γc and gene therapy w/o conditioning does not. Acute graft rejection: attack by transplant as antigens displayed in donors APCs (direct recognition) Chronic graft rejection: attack by transplant as antigens displayed on recipient APCs taken from the donors APCs (indirect recognition). APC-derived cytokines influence T cell differentiation:\ Naive t cells subsets are influenced by cytonkines and are characterised by the upregulated transcriptional factors. Th1: IL-12, T-bet/STAT4IFN-gdrive macrophages activity useful for pathogens/tumour Th2: IL-4, GATA3/STAT6 IL-5 IL-4IL-13 Eosinophils+IgE for allergy and extracellular pathogens Tfh: IL-6 IL-21 IL27,BCL6IL-21 B-cell + igG for Bcell help and memory. iTreg: IL-10/TGF-b/ Foxp3restricts T cell effect by producing IL-10/TGF-b Th17: IL-1 IL-23, RORgt/STAT3: IL17A IL17F(for neutrophiles) IL-22 IL-26(For antimicrobial peptides) fungi/bacteria protection. TCR structure: Alpha and beta chains which have 3 hypervariable(CDR 1,2,3) regions each, a constant domain, a transmembrane region and a cytoplasmic tail where CD3 zeta is found.CD3 signalling proteins consisting of two outer epsilons, gamma, delta and zeta on the tcr tail. MHC class I structure. A1&2 has 2-Alpha helical for the sides of peptide binding groove and 2-beta pleated sheets forming the base of the binding groove between a1&2 with 2 Ig-like domains, a3 and b2 MHC class II structure. A1 has 2-Alpha helical for the sides of peptide binding groove and one-beta pleated sheets forming the base of the binding groove with 2 Ig-like domain one a2 and one b2. HLA: HLA-B27 have survival to HIV and hepatitis C(viral infection) but increase autoimmunity such as reactive arthritis because it is identified as foreign. Gene therapy: Viral delivery: use DNA and RNA(Reverse transcriptase to form DNA) from Viruses. Viral delivery is more efficient and longer lasting but not reversible Non-viral delivery: liposomes get DNA into the cell or cell penetrating peptides directly puts a functional proteins and this options can be repeated and has the ability to titrate the dose. Editing gene techniques (e.g., CRISPR/Cas9) for modifying genes in cells ex vivo includes various nucleic acids like plasmid DNA, siRNA. Delivery is categorizes into single-class: For nucleic acid packaging, solid lipid Nps or polymeric NPs can be administered via the eye or gold NPs and dendrimers to the nose while hybrid systems with are a combination such as surfactant-phospholipid for organ administration like the gut. COVID has accelerated nucleic acid based therapeutic research: using RNA to be packaged and inserted to make a peptide that will be displayed by an APC. The vaccine in covid Fail to prevent transmission but was safe and had a higher success rate to traditional vaccines. RNA is edited instead of having uridine that can be recognised by TRL and degrade the RNA, it is substituted with N1-methylpseudouridine allowing it to be transcribed, It is now used to develop other vaccines and therapies such as caner. We have some that self replicate such as self-amplifying and trans-amplifying that have the enzyme replicase. and that are non-replicating. CLASS 2: **Vaccine adjuvants** Koch's postulates: microorganism identified at any stage of the disease, isolated and grown in pure culture, used to infect healthy host and cause the same symptoms then re-isolated to show the same as the first pure culture. Vaccine: material originating from a microorganism or other parasite that induces an immunologically mediated resistance to disease. Ideally administered as early as possible with long effect without ever observing symptoms/disease and Mucosally administered Herd immunity: Transmission of vaccine from vaccinated too non-vaccinated Herd protection: preventing of transmission of disease from the vaccinated. Types of vaccine.Whole/purified antigen Heterotypic: using a variant that gives less symptoms in humans, gotten from another animal host. Eg mycobacterium bovis for tuberculosis. Attenuated: An **attenuated vaccine** is created by weakening a pathogen (virus or bacteria) through processes like repeated culturing in non-human cells, genetic modification, or altering growth conditions. Eg Yellow fever. Live Attenuated can replicate and induce a response. Inactivated: destroyed by heat, radiation or antibodies. Eg Cholera Advantage: Whole virus is used so not just targeting a single antigen used for covid-19 as well. Subunits(purified antigen vaccine): surface protein or viral genetic material eg. hepatitis B. In covid-19. **Recombinant protein vaccines** use genetically engineered proteins from a pathogen to trigger an immune response. These proteins are produced in a lab, ensuring safety and specificity without using live pathogens. Conjugate: A **conjugate vaccine** links a weak antigen (usually a bacterial polysaccharide) to a strong carrier protein, enhancing the immune response and allowing TCR to also react to something else apart from a peptide. Eg influenza Toxiod: inactive toxic compound eg tetanus New gen vaccines: Recombinant proteins, Protein/polysaccharide conjugates, Synthetic peptides, DNA vaccines, RNA vaccines, Viral vectors. Some of the challenges are: Dictate the type of T cell response induced (e.g. CD4+ Th1, ThIL-17, CD8+ CTL , CTL is not useful for non-viral attacks. Adjuvants: Substances that can be incorporated into vaccines to enhance the magnitude, breadth and quality of the immune response to antigens. Important for mass production of vaccines during pandemics and produce Long lasting (memory). Types: Alum: effective th2 inducer but poor at CTL used un HepA&B vaccines liposomes(malaria, influenza): phospholipid vesicle vaccine based on viral membrane protein to exploit fusogenic properties. , Iscoms, squalene a lipid (influenza) and PLGA microparticles a biodegradable polymer, Depending on the size(circulation time and extravasation varies and either activates CD4 or CD8, T cells), shape(diffusion speed and binding),charge(internalized and degraded),material(reactive or opened up), surface(regulate expression or reprogramming) and cargo(remodel) Lipid particles: MF59(squalene oil-in-water emulsion) ASO3(squalene oil-in-water emulsion with -α-tocopherol (vitamin E),) liposomes(malaria, influenza): phospholipid vesicle vaccine based on viral membrane protein to exploit fusogenic properties immunostimulatory adjuvant: Monophosphoryl lipid A (MPL) :MPL is derived from the lipopolysaccharide (LPS) of **Gram-negative bacteria** LPS picked up by TLR4 and enhances Th1 and innate immune responses. Component of AS01 (with QS21 and liposomes) and AS04 (with alum) QS21: Induces Th1 responses and CTL in mice from bark of the tree *Quillaja Saponaria natural glycosides.* It is a fraction from Quil A but mixed with cholesterol reduces reactivity. Component of AS01 (with MPL and liposomes) and ISCOMS Unmethylated CpG: Activates TLR-9, Activate B cell, NK cell and monocyte/macrophages and DC, Induces Th1 responses including IgG2a, CTL responses. Unmethylated CpG is frequent in prokaryotes thus your TLR will indicate this as a foreign viral RNA to induce the immune response. It is a good vaccine booster. Viral vectors: Live vectors mimic natural infection e.g Adenovirus vectors, insert the foreign gene into them and they can carry it into the cells for replication with their own genes. Eg :**Vaccinia, NYVAC** is a weakened version of the vaccinia virus used as a carrier to deliver HIV antigens. It can also include genes for cytokines (called ALVAC) to enhance immune responses. Immunity to SARS-CoV-2: SPIKE protein binds to ACE2 receptor allowing the release of viral RNA.\ Antibodies that bind to S protein and especially to its receptor binding domain (RBD) prevent binding of the virus =neutralizing. Types of SARS-CoV-2 vaccines: Inactivated vaccine: grew cell in chemically inactive culture alum adjuvant added. Good because whole pathogen is the vaccine but takes a long time because they needed the actual various to start the process. Live attenuated vaccine: grown in a non-human cell culture and low temp to weaken the microbe and given nasally. Takes a long time and potential safety issues. Recombinant protein(subunit vaccine): S protein using viral vector with MF59 adjuvant, injected thus not a strong mucosal immunity(Trm). There is the replicative inactive vectors(S protein in another virus eg adenovirus whose replication is disabled. Replicate active vaccines: Measles vaccine expressing spike and can replicate, given nasally. DNA vaccines: electroporation induce uptake of vector. Plasmid containing s protein gene with mammalian expression promoters. Expressed in host tissue after injection. Very good and suitable for upscale but electroporating device needed for injection An ideal DNA vaccine should have strong promoter region, termination sequence, selective marker, good delivery: Gold particles are used as carriers in **gene guns** for delivering plasmid DNA into cells. In this process, DNA is bound to microscopic gold particles, which are then propelled into tissues (such as muscle or skin) using gas pressure RNA vaccines: and self-replicating RNA (SAM) vaccines: Delivered into cells using lipid nanoparticles. Challenge is that it should be kept frozen. Converting the Uridine to N1-methylpseudouridine so that is it not recognised by TLRs and RLRs. **CLASS 3: Functional analysis of genes by RNA interference** Initial indications came from plants: Overexpression of chalcone synthase an enzyme required for colour pigment production rather reduced the expression of the colour temporarily. This is said to have been RNAi because over expression of the RNA sequence caused RNA to overlap and make dsRNA which is cleaved into siRNA(small interfering RNA) that can, in the RNAi pathway, break down mRNA, leading to a knockdown. Basic Concept: Antisense mRNA can be used to generate mutants Effects: Vector DNA has two promoter regions starting the coding of the RNA but in the opposite direction. Sense RNA promoter region is T3 polymerase and antisense is T7 promoter for T7 polymerase. The sense and antisense strands hybridize (bind together), which prevents the translation of the sense mRNA into protein. This is crucial for gene silencing causing the same effect as a null mutation(gene does not function). Working more into this lead, is was also that RNA demonstrated that the control (sense strand injected only) also caused a loss of function. It was now known that ss-anti-RNA can cause the disfunction but ds-RNA also leads to the same effect as seen in the plant overexpression of chalcone synthase What is the mechanism of dsRNA? Experiments use the classical RNAi pathway. siRNAs (small interfering RNA) or anti-viral mechanism RNA virus or a transposon is in the process of copying itself produces dsRNA Classic RNAi pathway needs: Dicer, Small interfering RNA (siRNA), RNA-Induced Silencing Complex (RISC) Steps: Dicer enzyme, an RNase III endonuclease, recognizes and cleaves the dsRNA into small fragments of around 21-23 nucleotides called small interfering RNAs (siRNAs have 2- nucleotide 3' overhangs) having the guide strand(antisense) and passenger strand(sense) RISC is composed of multiple proteins, including a Slicer (Argonaute (Ago)), which plays a key role in RNA cleavage using the guide strand from the unwounded siRNA. The guide strand within RISC base-pairs with its complementary target mRNA, based on sequence complementarity. mRNA Degradation: Argonaute, as part of the RISC complex, cleaves the target mRNA, leading to its degradation. Major classes of small RNAs: siRNA: produced by cleavage of long endogenous or exogenous dsRNA or miRNA: noncoding regulatory RNAs that are single stranded that occasionally fold form ds regions(hairpins) by a complex consisting a(Drosha) and (Pasha), processed by Dicer to from dsRNA for incorporation into RISC. miRNA is used for different roles such as brain and heart development and is a regulatory RNA. Uses of dsRNA: interfering agent, Sequence-specific loss of mRNA and protein, Effects can cross cell barriers. Problem: Not all cells take up the dsRNA or siRNA, Problems with abundant proteins or proteins with low turnover(meaning it only downregulates not deltes thus sometimes only a small portion of protein is needed to continue the function even if 90% does not function) Conditional activation: Generate a cell line that express a T7 polymerase (to transcribe the target RNA) and a mechanism to control the polymerase usually a tetracycline operator: Tet on/off switch (transfection) with the induction of tetracycline. A marker to monitor if the gene was taken in by the cell. Introduce a construct where the target sequence GREEN (of gene you wish to knockdown) is inserted between flanking T7 promotors which drives transcription of the both strands(sense, anti-sense) double T7 promoters that flank the target sequence. Another method is to produce stem-loop (hairpin) structures which can be transcribed off a single T7 promotor: transcript fold s back to create stem/loop Analysis of phenotype: comparison of cells grown in presence and absence of inducer +/- tetracycline: Growth curve analysis for potential growth phenotypes 2) Morphological analysis, very often there are clear phenotyes, e.g. motility defects, abnormal shape etc 3) Confirm production of DS RNA/loss of target mRNA Northern blotting or QRT-PCR 4) Loss of protein requires some way to detect gene product: Antibody based Western blots/ELISA or direct assay for activity if known. 5) Controls for comparison extremely important: loading etc. 6) Important to monitor effects over time: phenotypes may be early or late in RNAi experiments e.g. protein stability. **Source of siRNA**: **Vector-based RNAi** uses plasmids or viral vectors to express short hairpin RNAs (shRNAs) that are processed into siRNAs within the cell, while **synthetic siRNA-based RNAi** involves the direct introduction of chemically synthesized double-stranded RNA molecules that are ready for incorporation into the RNA-induced silencing complex (RISC) without the need for transcription within the cell. Problems with siRNA: : lack of tight off control thus production of dsRNA in the absence of the inducer. Solution: check for production of dsRNA, better regulated system, target transcriptionally silent sites. OR affecting other genes and secondary knockdown of related proteins. Solution: Always check for sequence similarity in target region, check related mRNA levels as a control. OR These include stimulation of a subset of genes involved in the IFN response which can leads to effects on other targets. Presence of dsRNA can also lead to activation of TLRs VIVO RNAi: Transgenic RNAi: transgenic mice that express RNAi constructs, typically through the introduction of short hairpin RNAs (shRNAs) or other RNAi elements.  Local RNAi: Deliver RNAi constructs specifically to targeted tissues or cells.  Systemic RNAi: administration of RNAi agents (like synthetic siRNAs or direct dsRNAs) that circulate throughout the body and can target multiple tissues challenges for RNAi based therapeutics: size and structure prevents them from diffusing readily across membranes. elivery systems face issues with excipient toxicity, which can limit drug doses and cause harmful effects in nanoparticle formulations. break down of siRNA quickly, reducing its effectiveness in silencing target genes, matches between RNAi guide strands and non- targeted mRNAs the only way to ensure safety is via extensive testing ; some off- target RNAi effects may be unavoidable. Chemical modifications : essential for RNAi: attenuating activation of innate sensors that detect Nucleic acids/PAMPs dsRNA to resist degradation AND enhance antisense strand selectivity for RISC and reduce off- target RNAi Delivery systems under investigation: Naked siRNA Small \- molecule ligand conjugated to RNAi agent Lipid nanoparticles Known mechanisms: Patisiran consists of the small interfering RNA (siRNA) shown in complex with lipid excipients. The components are assembled under acidic pH into lipid nanoparticles (LNPs) and injected intravenously every 3 weeks at dosages of 0.3 mg per kg. The siRNA targets the 3ʹ untranslated region (UTR) of the TTR gene to silence all possible mRNAs with coding region mutations. RNA interference (RNAi) silencing results in sustained \>70% reductions of circulating TTR proteins, effectively stopping deposition of TTR TTR misfolding and aggregation is known to be associated with several amyloid diseases; senile systemic amyloidosis (SSA). Experiment to check if the insert and check the genome  Use PCR to amplify the region of interest (ORF/UTR).  Insert this amplified region into an RNAi vector and pick clones.  Prepare a large amount of the vector (Maxiprep).  Linearize the vector (cut it at a specific site like Not-1) for integration.  Transfect cells with the DNA using electroporation.  After 12-24 hours, apply selection using antibiotics (like Phleomycin or Hygromycin).  Expand the surviving clones.  Analyze the cells by growing them with or without an inducer (like tetracycline). **RNA Interference Overview** 1. **How was classical RNA interference and the significance of dsRNA discovered?** - The initial indications of RNA interference (RNAi) came from plant studies, particularly the overexpression of the chalcone synthase gene, which unexpectedly reduced color pigment expression. This phenomenon suggested that excess RNA caused overlapping sequences, resulting in double-stranded RNA (dsRNA) formation. This dsRNA was then cleaved into small interfering RNAs (siRNAs), which could degrade mRNA and lead to gene knockdown. 2. **Who were Fire and Mello?** - Andrew Fire and Craig Mello are scientists credited with the discovery of RNA interference. They conducted pioneering experiments demonstrating that introducing dsRNA into nematodes resulted in the specific silencing of genes, significantly contributing to the understanding of RNAi. 3. **What is Dicer?** - Dicer is an RNase III endonuclease that plays a crucial role in the RNAi pathway. It recognizes and cleaves long dsRNA into smaller fragments of approximately 21-23 nucleotides known as siRNAs. These siRNAs have 2-nucleotide 3' overhangs. 4. **What is the RISC?** - The RNA-Induced Silencing Complex (RISC) is a multiprotein complex essential for RNAi. It incorporates one strand of the siRNA (the guide strand) and facilitates the base pairing of this strand with complementary target mRNA, leading to its degradation by Argonaute, a key protein within RISC. 5. **What are the key features of a small interfering RNA?** - Small interfering RNAs (siRNAs) are typically 21-23 nucleotides long, have 2-nucleotide 3\' overhangs, and are derived from the cleavage of long dsRNA. They are critical for the RNAi mechanism, mediating sequence-specific degradation of target mRNA. 6. **miRNA vs. siRNA:** - **miRNA (microRNA)**: Noncoding regulatory RNAs that are usually single-stranded and can form hairpin structures. They play roles in regulating gene expression and are processed by Dicer. - **siRNA (small interfering RNA)**: Typically derived from long dsRNA, siRNAs are primarily involved in the degradation of complementary mRNA and are essential for the classical RNAi pathway. 7. **What are the key features of RNAi? Why is it useful?** - RNA interference allows for specific and sequence-dependent gene silencing. Key features include the ability to target and degrade specific mRNA, cross-cell barrier effects, and potential therapeutic applications. It is useful for functional genomics, gene knockdown studies, and developing RNAi-based therapies. 8. **How would you perform an RNAi experiment?** - To perform an RNAi experiment, design and synthesize specific siRNAs targeting the gene of interest. Introduce these siRNAs into the cell using transfection methods. Assess the knockdown efficiency by measuring levels of mRNA (using qRT-PCR) and protein (using Western blotting or ELISA). 9. **What do we mean by vector-based and siRNA-based approaches?** - **Vector-based RNAi**: Utilizes plasmids or viral vectors to express short hairpin RNAs (shRNAs), which are processed into siRNAs within the cell. - **siRNA-based approaches**: Involve the direct introduction of synthetic siRNAs into the cell, which are ready to incorporate into RISC without the need for transcription. 10. **What is the difference between opposing T7 and hairpin-based RNAi?** - **Opposing T7 RNAi**: Involves using two promoter regions (T7) in opposite directions to generate dsRNA from sense and antisense strands. - **Hairpin-based RNAi**: Involves designing RNA constructs that form a hairpin structure, allowing a single promoter to drive transcription and subsequent processing by Dicer. 11. **What is conditional RNAi, and how does Tet on/off work?** - Conditional RNAi involves a system where the expression of RNAi constructs can be controlled, such as the Tet on/off system. In this method, a tetracycline operator regulates T7 polymerase activity, allowing for controlled transcription of the target RNA in response to tetracycline presence. 12. **Lentivirus and RNAi in mammals: why use a virus?** - Lentiviral vectors can efficiently deliver RNAi constructs into mammalian cells, allowing for stable integration and long-term gene silencing. Their ability to infect dividing and non-dividing cells makes them a powerful tool for RNAi-based therapies. 13. **Could you describe an RNAi experiment?** - In an RNAi experiment, you would design specific siRNAs targeting a gene of interest. Introduce these siRNAs into cultured cells via transfection. Monitor the effects by assessing mRNA and protein levels through qRT-PCR and Western blotting, respectively, comparing treated versus control groups. 14. **Knockdown vs. knockout:** - **Knockdown** refers to the reduction of gene expression through RNAi, which is often temporary and does not completely eliminate the gene's function. - **Knockout** involves the complete and permanent loss of gene function, typically through genetic engineering techniques. 15. **How would you assess whether an RNAi experiment is working? Levels of mRNA, protein, etc.?** - Assess RNAi efficacy by measuring target mRNA levels using qRT-PCR and protein levels using Western blotting or ELISA. Additional methods include Northern blotting to confirm dsRNA production. 16. **What are the difficulties with RNAi? How can I be certain that I have no off-target effects?** - Challenges with RNAi include off-target effects, incomplete knockdown, and the potential for activating immune responses. To minimize off-target effects, perform thorough sequence analysis of target regions and use multiple siRNAs targeting different regions of the same gene for validation. 17. **Any idea about RNAi libraries?** - RNAi libraries consist of collections of siRNAs targeting a wide range of genes, allowing for high-throughput screening to identify gene functions and potential therapeutic targets. 18. **RNAi therapies?** - RNAi therapies aim to treat diseases by specifically silencing disease-causing genes. Examples include Patisiran, which uses lipid nanoparticles to deliver siRNAs targeting the TTR gene, reducing circulating TTR protein levels in patients with amyloid diseases. **Class 4** Types of Viral vectors: adenovirus : dsDNA AAV: ssDNA Retrovirus incorporation: Virus is taken in then , uncoated, viral rna and goes through reverse transcription to make DNA and integrase is used to integrate it into DNA. Prevent HIV infection: receptor CCR5&CXCR4 are used to bind to the HIV, using the CRISP9 technique, cytosine is added to introduce the stop codon within the sequence or remove the start codon(the UTR 3' and 5' respectively). Thus the receptors are not made and HIV cannot bind. To make a therapy for SCID some safety precautions needs to be taken: remove the LT3 promoter and inactivated U3 to stop replication of Virus and integrate directly To specific site while adding a suicide gene code. Lentiviral(**Lentiviral** refers to a type of virus derived from the retrovirus family) gene packaging: In the virus the Viral genome is on a separate DNA strand to that of the therapeutics DNA strand. Sickle cell disease -- β-thalassemia: sickle cell hemoglobin forms long chains cloating red blood cells together leading to iron overload. Treated by bone marrow transplant or blood transfusion. AAV transduction: enters in the same format at when B cells get an antigen. The virus can also espcase the lysosome and enter into the nucleus for self replication and integrating its DNA into the host cell. How is AAV produced: Plasmid Transfection: Three types of plasmids are introduced into producer cells (often HEK293 cells): Helper plasmid: Contains genes from adenovirus essential to act as a virus Rep/Cap plasmid: Encodes AAV replication (Rep) and capsid (Cap) proteins, which are necessary for viral assembly. Transgene plasmid: Contains the gene of interest flanked by AAV\'s. Cell Culture and Transfection: The plasmids are co-transfected into HEK293 and culture. The AAV is now separated by centrifugation in a 60% Iodixanol solution with different density solution by different iodixanol percentage. As the centrifugation occurs,the AAV supernatant will divide it self depending on there size and weight and density a AAV at the second to bottom.You can no puncture and allow the AAV to drip out for collection. In terms of coating selection to make sure the virus in engulfed, you can find naturally occuring AAV or design one or create new receptors to help in the engulfing. PRR are able to recognise the virus and the RNA inducing immune response to kill the viruses or kill the cell infected. TLR, RLR and etc. In practice, chimpanzees AAV are used to carry spike protein for the vaccines for COVID. A muscle disorder in the dystrophin causing A shortening of the intracellular protein. Becker muscle dystrophy is the shortening by a mutation in one of the exons coding for an amino acid, there are about 79 exons that match each other and if one deleted, but the chain can continue, the protein can be made but just short, DMD is caused when the chain cannot continue thus causing no protein to be formed. For the therapy we use antisense DNA to skip the mutated Exon that causes the DMD thus allowing it to create the protein but just shorter but still functional. Once produced, AAV vectors can be used for delivering genes to target cells or tissues in research and therapeutic applications. How is DNA inserted. CRIP-CAS: guide DNA binds to specific site and cas9 cleaves it. Very short time to make but could bind to the wrong effector. ZFN (Zinc Finger Nucleases): ZFNs are engineered proteins that use zinc finger domains to bind specific DNA sequences and a FokI nuclease domain to cut DNA at the target site. Once the DNA is cut, the cell repairs the break, allowing for gene editing.Takes long to make but very good TALEN (Transcription Activator-Like Effector Nucleases): TALENs use TALE proteins to recognize specific DNA sequences and a FokI nuclease to cut the DNA. This double-strand break triggers cellular repair, enabling precise gene modifications. Shorter time and quite precise. DNA Repair: Once the DNA is cut, the cell\'s natural repair mechanisms are activated: Non-Homologous End Joining (NHEJ): The break is repaired by directly joining the broken ends. This method is prone to errors and often results in small insertions or deletions, leading to gene disruption or knockout. Homology-Directed Repair (HDR): If a repair template (a piece of DNA with sequences similar to the broken region) is provided, the cell can use it to repair the break accurately, allowing for precise insertion, deletion, or modification of DNA. First ever CRISPR-CAS9 editing: Leber congenital amaurosis type 10 is a rare inherited blindness caused by an intronic mutation in the CEP290 gene, leading to faulty splicing and loss of primary cilia in photoreceptors. Editas and Allergen are developing EDIT-101 (AGN-151187) gene therapy using an AAV5 vector to deliver two guide RNAs and the Cas9 enzyme, aiming to restore CEP290 protein production specifically in photoreceptors. Barriers to genome editing component: Proteins, RNA and DNA are packed in nanoparticles vehicles which can be subject to phagocytosis, degradation and extraction. They can also induce an immune response. Methods of delivery: In vitro nucleus cell injection(cells in the culture are injected release the genomic information in the nucleus) or injecting the Embryo and electroporation. **CLASS 5 CART-T** - **Tumor-Associated Antigens (TAAs) these are Public Antigens**: These are antigens that are commonly shared among different tumors or patients. - **Overexpressed Antigens**: These are proteins that are present at higher levels in tumor cells compared to normal cells. Examples include Her2/neu, BCR-ABL, and survivin. - **Tissue Differentiation Antigens**: These are proteins that are typically expressed in specific tissues but may be overexpressed in tumors. Examples include Gp100 and Melan-A. - **Tumor Germline Antigens**: These are antigens that are normally expressed in germ cells but can also be found in tumors. Examples include MAGE, BAGE, and GAGE. **Tumor-Specific Antigens (TSAs)** - **Oncoviral Antigens are Shared Antigens**: These are antigens that may be found in multiple tumor types but are not universally present: These are antigens derived from viruses that can cause cancer, such as HPV E6 and E7, and EBV. Cancer treatments are only associated with membrane proteins to help immune cells identify them but there are about 70% intracellular proteins that cannot be targeted. Autologous tumor-infiltrating lymphocytes (TILs): Cells are extracted from tumour site and separated and cultured. They are now tested for tumour recognition and the ones that can are selected and cultured for more to be produced then reinfused into the patient. Before reinfusion lymphoids should be depleted using chemicals to insure TILs survive (nutrients and etc). Exploiting T cells ability to recognise tumor-specific mutations: Tumour cells DNA are extracted and sequenced to find the mutation, the mutated peptide is sequenced and displayed on an APC to be identified by T cells which will be isolated and expanded and injected into patient. T cells from blood are inserted with Viral or non-viral gene. T cells and CAR-T cells that can bind to an antigen of the tumour cell are expanded and injected with IL-2 to the patient. CAR-T cells (chimeric antigen receptor- T cells). Structure: Variable light chain, and heavy chain hinged to transmembrane domain,in the cytosol there is a co-stimulatory molecule and then a signalling domain CD3 zeta. Comparison: 6 ITAMS, no MHC restriction required and recognise surfaceome. Universal Car-Ts (UCAT19) forming: Insert CAR, CD 20 and suicide gene using TALLENS. And KO the T cell alpha, PD-1 and CD52 protein. It can be used to attack Tumour cells with CD20 administrated via bone marrow. CAR- Tcell therapy: Cell Collection: Blood is drawn from the patient to collect T-cells. T-cell Modification: The collected T-cells are sent to a lab where they are genetically modified to express a chimeric antigen receptor (CAR) that targets cancer cells. Cell Expansion: The modified T-cells are then expanded in the lab to increase their numbers. Infusion: The expanded CAR T-cells are infused back into the patient through an intravenous (IV) line. Targeting Cancer Cells: The CAR T-cells seek out and bind to cancer cells, leading to their destruction Mood of action: **Direct Cytotoxicity**: CAR T cells recognize and bind to specific antigens on the surface of antigen positive-tumor cells activating granzyme and perforin for cytolysis. **Cytokine Release**: AR T cells secrete pro-inflammatory cytokines like IFN-γ and TNF-α, which enhance the immune response, recruit other immune cells, and create an anti-tumor microenvironment.. **Immune Modulation**: **Fas-FasL Interaction (Apoptosis Induction)**: CAR T cells express Fas ligand (FasL) on their surface, which binds to the Fas receptor on tumor cells, triggering the apoptotic pathway and leading to tumor cell death through programmed cell death (apoptosis). Problems with CAR T treatment: Neurotoxicity from all the cytokines causing seizures or organ dysfunction, Tumour cells can down-regulate CD20 or tumour displaying similar antigen to another functioning cells thus making it invisible to CAR -T cells. Cannot harvest enough T cells to convert to CAR-T cells for the treatment. safety mechanisms: Unlike traditional CAR-T cells, which are constantly active once introduced, GO-CAR-T cells use a \"switch\" mechanism like MC that turns on CAR when rimiducid is bound. Innovations in T cell therapy: Gene editing via CRISPR allows for precise modifications in T cells, such as enhancing their anti-tumor activity or eliminating inhibitory checkpoints like PD-1. (Tregs) to express chimeric antigen receptors (CARs) that specifically target certain antigens. These modified Tregs can suppress immune responses making then useful for organ transplant. DCs are collected, exposed to cancer antigens in the lab, and then injected back into the patient. This process helps the immune system recognize and attack cancer cells more effectively. In vivo tissue engineering: three-dimensional framework or structure that supports the growth and organization of cells into functional tissues by using biomaterials, stem cells, and growth factors to stimulate the body\'s natural repair processes In vitro tissue engineering involves creating tissues in a lab setting. It begins with isolating cells from a donor, which are then cultured on a biodegradable scaffold that supports cell growth. The cells are kept in controlled conditions with optimal nutrients and oxygen, and growth factors may be added to guide their development. Over time, the cells proliferate and organize into functional tissue structures, which are evaluated for their potential therapeutic applications.