Gene Therapy Scheme PDF

✍🏼 Scheme Index Bold = name of drug Underlined = clinical trial Italicize = approved product Red colour = gene therapy Green colour = cell therapy Blue colour = gene editing...

✍🏼 Scheme Index Bold = name of drug Underlined = clinical trial Italicize = approved product Red colour = gene therapy Green colour = cell therapy Blue colour = gene editing Purple colour = type of viral vector Orange colour = stem cells Pink colour = therapeutic nucleic acids 1. Genetic engineering Advanced therapy medicinal products (ATMPs) 1. Gene therapy medicines: insert a recombinant gene (stretch of DNA created in the lab by bringing together DNA from different sources) that lead to a therapeutic, prophylactic or diagnostic effect 2. Somatic-cell therapy medicines: cells or tissues changed in their biological characteristics to cure, diagnose or prevent diseases 3. Tissue-engineered medicines: cells or tissues modified to repair, regenerate or replace human tissue Genetic engineering applications 1. Recombinant proteins: since genetic code is universal, proteins can be produced in cells in which gene is not initially present Process: Gene isolated and inserted in a plasmid (circular DNA) Obtain a recombinant DNA used to transfect a bacterium Recombinant bacterium has gained an ability encoded by the gene inserted → protein production Example: Gleevec blocks the ATP binding-pocket of the kinase BCR-ABL obtained by the translocation of chr 9 and 22 (Philadelphia chr) responsible for the CML (Chronic Myelogenous Leukemia) → it has substituted ribozyme use 2. Genetically-modified plants (GMOs) 3. Genetically-modified animals: transfer the gene of interest into a transgenic animal to understand aspects of gene function and development 4. Gene therapy: treatment for genetic disorders which involve adding a normally-functioning copy of the gene to restore normal function Types: Germiline gene therapy: permanent transfer of a gene into sperm or egg cells → future generations will be cured Somatic cell gene therapy: transfer of genes to affected cells and correction of deleterious effects via long-term integration → only one approved Vectors: carriers for gene transfer Direct uptake of oligont or plasmid / low efficiency and rare Scheme 1  Chemical metods: liposomes Protein-DNA complexes DNA-complexed nanoparticles Physical methods: electroporation, gene gun Viral vectors: Adenovirus Adeno-associated virus Retrovirus (e.g. murian/avian virus) Lentivirus (e.g. HIV-1) Applications: replace a missed function Single-gene disorder Recessive diseases X-linked inherited Techniques: In-vivo: delivery of new genetic material directly to target cells within the body Ex-vivo: target cells are removed from the body and then genetically-modified Diseases: Scheme 2  Adenosine Deaminase (ADA) deficiency: autosomal recessive immunodeficiency → therapy Strimvelis: autologous CD34+ BM cells transduced with retroviral vector containing ADA gene into children with ADA-SCID Leber’s Congenital Amaurosis (LCA): retinal degeneration due to recessive mutations in RPE65 gene → therapy: subretinal injection into retinal pigment epithelial (RPE) cells of AAV2-hRPE65 5. Gene editing: type of genetic engineering in which DNA is inserted into site-specific locations through the action of programmable nucleases that recognise genomic loci and induce DSBs then repaired either through NHEJ or HDR Programmable nucleases: Meganucleases Zinc finger nucleases TALEN CRISPR/Cas9 Gene editing therapy approved: sickle cell disease 2. Ageing, geroscience and nutritional anti-ageing strategies Ageing 💡 Ageing: progressive, generalized impairment of function, resulting in increasing vulnerability to environmental challenge and a growing risk of disease and death. Changes in the organism: Diminished regeneration and proliferation capacities of both somatic and embryonic cells Increased cross-linking of extra-cellular matrix → rigidity of tissues Increased macromolecules accumulation → accumulation diseases Increased number of glycosilation products Theories of ageing: 1. Free radical theory: damage produced by free radicals, glucose or other agents slowly disrupt cellular macromolecules → age-related increase in somatic mutations and other forms of DNA damage which induce activation of DNA damage response (ATM proteins) Scheme 3  2. Mitochondrial theory: mtDNA mutations might lead to increase in ROS production and gradual energy decline isp-1 mutant → longer-lived clk-1 mutant → longer-lived mev-1 mutant → shorter-lived Indy gene → increase life span Mth gene → increase life span Mutations which make mitochondrion less functional and thus able to generate lower amount of ROS account for increased life span. 3. Telomere loss theory: decline in cellular division capacity with age linked to progressive shortening of telomeres as cells divide (S phase of cell cycle) which is recognised as a damage 💡 Telomeres: long stretches of DNA with repetitive sequences at the end of chr covered by proteins which wrap around them to maintain genome stability. 4. Altered proteins theory and waste accumulation theory: accumulation of damaged proteins (Alzheimer’s, Parkinson, etc) 5. Antagonist pleiotropic theory 6. Mutation accumulation theory: de novo germiline mutations not efficiently selected by natural selection over successive generations which accumulate within the genome 7. Rate of living theory: metabolic rate is inversely correlated with longevity → smaller mammals tend to have high metabolic rates and thus tend to die earlier / opposite situation withtin a specie 8. Weak link theory: systems particularly weak over time → dysfunctions accelerate senescence of whole organism 9. Error catastrophe theory: errors in DNA transcription or RNA translation leading to genetic errors 10. Master clock theory: ageing under genetic control 11. Disposable soma theory 12. Combined network theory: multiplicity of ageing mechanisms Organisms associated with longevity: C. elegans Turtles, deep water fishes and americal lobsters Big ban animals and salmons Hydra Biomarkers of ageing: Loss of proteostasis Mitochondrial dysfunction Oxidative damage Cellular senescence Epigenetic alterations: Nutrition and insuline methylation → decrease cell growth, proliferation and metabolic activity associated with longevity Sirt1 epigenetic regulator: deacetylation of histones and TFs associated with longevity (Resveratrol: activator of Sirt1 which works by activating AMPK pathway of autophagy) Scheme 4  Anti-ageing therapies: Antioxidants and free radical scavengers: modest effect Resveratrol and AMPK activators Rapamycin and mTOR inhibitors Statin Senolytic drugs: Dasatinio, Quercitin Metformin: first-line oral treatment for T2D which may decrease ageing Pre-clinical trial in monkeys: Reduce periodontal bone loss Higher accuracy in retriving food Restore thickness of frontal cortex (learning, memory) Decrease senescent cells, fibrosis and inflammatory cells Rescue expression of genes associated with inhibition of ageing-activating / activation of ageing-inhibiting pathways Rescue of ageing clocks Rescue neuronal cell types Induce neural differentiation toward post-mitotic neurons and increase Nrf2 phosphorylation (antioxidant response) TAME: ongoing Anti-inflammatory agents siRNA and microRNAs Dietary restriction: transcriptional and epigenetic changes Geroscience and nutritional anti-ageing strategies Anti-ageing nutritional strategies: 1. Caloric restriction: Strategies: 30% CR prolong life in warms, flies, yeast and mice 25% CR in humans (CALERIE) Benefit: Lower body temperature Lower blood glucose and insulin Reduce fat and weight Improve immune function decreasing cancer and infection (limit: decrease of proteins) Increase response to external stress Controversies: May improve survival because of its effect on weight loss May impact brain integrity if BMI120h: ketosis steady state and protein conservation phase (rely on autophgy) Benefit: Decreased chronic inflammation Improved insulin sensitivity Positive effect on gut microbiome Reduced blood pressure Controlled body weight and abdominal fat Controversies: Important muscle loss Rodents start to use muscle → protein conservation phase Less efficient compared to CR Progressive change from CR to increase calories lead to rebound effect Not preventive therapy but only indication for some patients 3. Time-restricted eating: Intermittent fasting: train the organism to rely on gluconeogenesis (16h metabolic switch) without entering in ketosis but remaining in protein preservation phase Periodic fasting: 1-2 days of fasting every week of ad-libitum diet Fasting Mimiking Diet (FMD): plant-based, micronutrient-rich, low-calorie, high-fat diet able to reduce biological age and disease risk independently from weight loss / need more data Therapeutic fasting (>36h): reach protein conservation phase and autophagy 3. Gene therapy Therapeutic nucleic acids 1. Protein-coding cDNAs: a. Proteins replacing missing cellular functions → application for monogenic diseases: Immunodeficiencies: ADA-SCID Haemophilia Leber’s congenital amaurosis Muscular dystrophies Cystic fibrosis Lysosomal storage diseases b. Proteins modulating cellular functions c. Proteins regulating cell survival d. Proteins activating IS e. Antibodies: anti-tumoral vaccination → delivered with viral vectors or RNA f. Growth factors: neurodegenerative and traumatic disorders Scheme 6  2. Small non-coding DNAs and RNAs: a. Antisense oligont: i. 1st gen: phosphorotioate ii. 2nd gen: MOE, LNA iii. 3rd gen: morpholino, PNA b. Ribozymes and DNAzymes: catalytic activity c. Regulatory RNAs (siRNAs, shRNAs, microRNAs): Hereditary multifactorial disorders: Familial hypercholesterolemia (FH) → target gene: apoB Age-related macular degeneration (AMD) → target gene: BEGF, VEGFR1, RPT801 Lateral amyotrophic sclerosis (LAS) → target gene: SOD1 Alzheimer’s disease (AD) → target gene: tau, APP Parkinson’s disease (PD) → target gene: synuclein Cancer: CML → target gene: BCR-Abl Infectious diseases d. DNA/RNA decoys and aptamers: RNA decoys: ssRNA able to bind microRNAs used as defense mechanism for mRNA Aptamers: nucleic acid-based (ssDNA or ssRNA) mAb which bind with high affinity and specificity to their target protein Viral vectors 1. Retroviral and lentiviral vectors: Classes of retrovirus: Orthoretrovirinae: alpharetrovirus, betaretrovirus, gammaretrovirus, deltaretrovirus, epsilonretrovirus, lentivirus (e.g. HIV-1) Spumaretrovirinae: spumavirus Genetic organization of retrovirus: Three genes: gag: structural and nucleic acid binding pol: protease and integrase env: envelope LTR: stretch of 400-700nt with promoter activity formed after integration of the virus Psi signal: packaging element which allows for packaging of retroviral RNA genome into viral capsid during replication Genome is used to generate proteins and retro-transcribe genome to generate mRNA to be incapsidated into viral particles depending on capsid signal. Scheme 7  Tumorigenic retroviruses Types: RSV (Rous Sarcoma Virus): expression of viral oncogenes (src) → fast-developing tumour MLV (Moloney-Murine leukemia virus): insertional mutagenesis and activation of cellular oncogenes → slow- developing tumour Mechanisms of tumorigenesis: Brings or activate an oncogene Disactivate and oncosuppressor Produce proteins able to mutate DNA Retroviral vectors: gag: Psi sequence: needed for encapsidation tRNA: primer for RT LTR: needed for transcription Therapeutic gene Production retroviral vectors: plasmid containing retroviral vector genome is transfected with Ca-P co-precipitation in packaging cells which stably express gag, pol, env Recombinant viral particles can enter target cells has they have envelope, they are retro- transcribed and integrated byt they encode for transgene only → never produce wt virus. Scheme 8  Pseudotyping: use of specific env to increase efficiency of vector to target specific cells (broader tropism) Scheme 9  Lentiviral virus → genetic organization of lentivirus: gag, pol, env Accessory genes → long mRNA encoding for 9 proteins: use of Rev protein able to bind RRE (Rev response element) allowing for active transfer of unspliced long transcripts in cytoplasm Scheme 10  Lentiviral vectors → genetic organization: First gen: 3 plasmids, 1 packaging plasmid tet, rev on a single plasmid gag, pol on a single plasmid Safety concerns: Recombination during manufacturing may generate RCL (replication-competent lentivirus) with even a broader tropism Recombination with wt virus in HIV+ subjects Lentiviral vector mobilization by wt virus Scheme 11  Second gen: 3 plasmids, 1 packaging plasmid tet, rev on a single plasmid gag, pol on a single plasmid Third gen: 4 plasmids, 2 packaging plasmids tet absent, rev on a separate plasmid gag, pol on a single plasmid Deletion in 3’ LTR-SIN: during RT they intrinsically inactivate themseleves by interfering with syntesis of wt LTR Scheme 12  Must pick a promoter to complement SIN deletion Reduce trans-activating activity of LTR which may activate oncogenes near integration site Characteristics: Integrate into host cells Transduce cells in active replication Are immunogenic Adv and disadv lentiviral vectors: Scheme 13  Differences between retroviral and lentiviral vectors: Retroviral vectors enter only when cells divides wherease lentiviral vectors have an active transport system which allows to enter in non-dividing cells Retroviral vectors integrate preferentially in hotspot MECOM wherease lentiviral vectors do not have preferences (less risk of leukemic events) Lentiviral vectors, when injected in-vivo, infect also non-dividing cells ending to be inactivated by complement → better application for ex-vivo gene therapy (especially epithelial) 2. Adenoviral vectors: Genetic organization: dsDNA Types: First gen: Adv: Wide repertoire of target cells (natural tropism for a variety of cells) Capacity of infection of both proliferative and quiescent cells High efficiency of transduction in-vivo Remain episomal Disadv: Difficult to administer because of IS response: high inflammatory response Transient expression Limited cloning capacity (