Hunter Syndrome and Gene Therapy

Hunter Syndrome Pathophysiology

• Hunter syndrome is a chronic progressive disease caused by the accumulation of partially degraded GAGs, leading to thickening of tissue and compromising of cell and organ function over time.
• The IDS gene produces the I2S protein, which is necessary for the breakdown of GAGs in lysosomes.
• Mutations in the IDS gene cause Hunter syndrome.
• Incorrect glycosylation of the I2S protein may interfere with lysosomal targeting through impaired binding to the mannose-6-phosphate receptor.

Enzyme Replacement Therapy (ERT)

• ERT with idursulfase (Elaprase) improves somatic signs and symptoms of Hunter syndrome.
• However, the drug does not cross the blood-brain barrier, making treatment of neurological aspects of the disease challenging.
• Intracerebroventricular injection is an option, but it poses difficulties for widespread use.
• Elaprase works by reducing the amount of creatinine in urine, indicating improved kidney health.

Gene Replacement Therapy

• Gene therapy involves delivering a functional IDS gene to the liver using AAVs.
• The IDS gene is inserted into the albumin gene, allowing for the production of the I2S protein in hepatocytes.
• The I2S protein facilitates the breakdown of GAGs in lysosomes, potentially reducing the burden of Hunter syndrome on hepatocyte cells.

AAV Vectors

• AAVs are single-stranded DNA viruses with a simple genome packaged in an icosahedral capsid.
• The genome is typically "gutted" to make room for the gene of interest, ensuring safety and efficiency.
• The size of the insertion is a limiting factor, and AAV vectors lack the integration-promoting gene, making them safe for gene therapy.

Gene Editing

• Gene editing involves modifying genes using site-directed nucleases such as ZFNs, TALENs, and CRISPR/Cas9.
• These nucleases introduce double-strand breaks, which can be repaired using homology-directed repair (HDR) or non-homologous end joining (NHEJ).
• HDR is a precise method, but NHEJ can result in insertions or deletions of nucleotides, disrupting the reading frame of genes.

Volume of Distribution

• Volume of distribution (Vd) is a theoretical concept that relates the plasma concentration to the total amount of drug in the body.
• Vd is affected by the drug's ability to move out of the plasma compartment, with lipophilic molecules having a larger Vd.

Metabolism and Excretion

• Metabolic degradation and renal excretion of unchanged active drugs are important factors in drug elimination.
• Renal clearance is the sum of glomerular filtration, tubular secretion, and reabsorption.
• Drug metabolism makes lipid-soluble drugs more water-soluble, allowing for excretion via the kidneys.
• Phase I reactions involve functionalization, making the drug more polar, while Phase II reactions involve conjugation with endogenous molecules.### Phase I Drug Metabolizing Enzymes
• Broad substrate specificity, meaning one enzyme can catalyze the metabolism of many different drugs
• One drug can be metabolized by multiple enzymes/isozymes
• Liver drug metabolizing enzymes are usually embedded in endoplasmic reticulum (microsomal)
• Important family of enzymes is cytochrome P450 "super" family, which catalyzes oxidative metabolism of a range of xenobiotics

Cytochrome P450s

• Differ in amino acid sequence, sensitivity to inhibitors and inducing agents, and substrate specificity
• Enzyme reaction requires molecular oxygen, NADPH, and NADPH-P450 reductase (a flavoprotein)
• Forms a hydroxylated product
• There are approximately 74 CYP450 gene families, with three major families involved in drug metabolism in the liver (CYP1, CYP2, CYP3)

Metabolising Enzymes

• Are potentially saturable, and activity can be influenced by many factors, including genetics, other drugs, and environmental factors
• Caution is needed when elimination involves enzymic metabolism, as drug interactions can occur via metabolizing enzymes

Drug Interaction via Metabolizing Enzymes

• Enzyme inhibition: drugs metabolized by the same enzyme, if administered together, will compete for the metabolizing enzyme
• Enzyme induction: some drugs can increase the activity/levels of the enzymes that metabolize them (e.g. alcohol, barbiturates)
• Interaction can also occur between drugs and "herbal" medicines or food components (e.g. St John's Wort, grapefruit juice)

Biliary Clearance of Drugs

• Enterohepatic recycling can occur, where drugs are excreted into the bile and then reabsorbed into the bloodstream

Other Routes for Excretion of Drugs

• Lungs can remove gases and volatile liquids (e.g. many general anaesthetics)
• Sweat, saliva, mucous, tears, and milk can excrete small amounts of drugs (some drugs in milk can be dangerous to infants)

Polymorphisms in Cytochrome P-450

• All genes encoding CYP1/2/3 family of P-450 enzymes are polymorphic
• Clinically important polymorphisms are seen in CYP2C9, CYP2C19, CYP2D6, and CYP3A4 (account for 60-70% of phase 1 dependent metabolism of clinically important drugs)
• Variations in CYP alleles can affect drug metabolism, e.g. CYP2D6 metabolizes 25% of drugs

Consequences of Polymorphisms in CYP2D6

• Poor metabolizers (no CYP2D6 enzymes) have drug metabolism that is too slow, leading to high drug levels and increased risk of adverse effects
• Ultrafast metabolizers (CYP2D6 gene duplications) have drug metabolism that is too fast, leading to no drug response at ordinary dosage

Warfarin

• Has a high rate of adverse events
• Warfarin maintenance doses are characterized by large inter-individual variability
• Warfarin pharmacogenetics: metabolism of warfarin occurs through CYP2C9, and clinically relevant SNPs have been identified in CYP2C9, resulting in reduced enzymatic activity

Pharmacogenomics

• The use of genetic information to guide drug therapy
• Differences in the response of individuals to drugs can be predicted from knowledge of genetic makeup

Pharmacokinetics

• The rate of elimination is driven by Cp (concentration of the drug in the plasma)
• Constant/repeated dosing will achieve a steady state Cp
• Time to reach steady state is determined by t½ (plasma half-life)
• Time for "removal" is determined by t½

Systemic Lupus Erythematosus (SLE)

• A systemic disease, mostly in women, with high morbidity
• Different epidemiologic subgroups (e.g. race/ethnicity, gender, and age of onset) tend to have varying degrees of disease activity and may thus affect disease outcome
• Disease manifestations include lupus malar "butterfly" rash, arthritis, CNS inflammation, and lupus nephritis
• Lupus nephritis is caused by deposition of immune complexes in kidneys, leading to injury to the glomerulus

Mortality in SLE

• Linked to early organ damage
• Survival probability in patients with and without early damage is affected by the presence of early damage

Autoantibodies in SLE

• Against molecules in nuclei, such as dsDNA, ribonucleoproteins, and other nuclear proteins complexed with nucleic acids
• These autoantigens are ligands for "danger sensors" on antigen-presenting cells and B cells

Genetic Predisposition

• Clearance of apoptotic particles and immune complexes
• The role of SLE risk alleles in the pathogenesis of SLE
• Polymorphisms in genes involved in the immune clearance of apoptotic particles and nucleic-acid-containing immune complexes
• Polymorphisms in genes involved in innate immunity and adaptive immunity

Environmental Triggers

• UV light, smoking, air pollution, trace elements, diet, oestrogens, hormone replacement, and medications

• Localized tissue damage, leading to release of autoantigens and localized inflammation, causing up-regulation of co-stimulatory proteins on antigen-presenting cells### Systemic Lupus Erythematosus (SLE)

• All components of the innate immune system are involved in the inflammatory response in SLE.

• Decreased clearance of apoptotic cells leads to prolonged exposure of potential auto-antigens, promoting tissue damage and pro-inflammatory cytokine production.

• Decreased anti-inflammatory heme oxygenase-1 (HO-1) and increased pro-inflammatory cytokine production contribute to the inflammatory response.

• Infiltrating tissue lesions and reduced number of ILC2 cells lead to increased pro-inflammatory cytokine production.

Innate Immune System Activation in SLE

• Activation of the innate immune system leads to increased tissue damage and pro-inflammatory cytokine production.
• Decreased clearance of apoptotic cells and decreased anti-inflammatory HO-1 contribute to the inflammatory response.

Lupus Nephritis

• Deposition of immune complexes in kidneys leads to glomerulonephritis.
• Immune complexes activate complement and lead to inflammation and tissue damage.

Cyclic Exacerbation of Disease

• Cyclic exacerbation of disease leads to irreversible tissue damage.
• Clinical disease onset is characterized by rash, nephritis, arthritis, leukopenia, and CNS inflammation.
• Irreversible tissue damage can lead to organ failure, such as renal failure, atherosclerosis, pulmonary fibrosis, and stroke.

Current Treatment for Autoimmune Diseases

• B cell-targeting therapies, such as anti-CD20 and anti-CD19 monoclonal antibodies, are used to deplete B cells.
• Monoclonal antibodies targeting B cell surface markers, such as rituximab and ocrelizumab, are approved for autoimmune diseases.
• Belimumab, a human IgG1 recombinant monoclonal antibody anti-BAFF, is approved for SLE.

Treatment Under Investigation for Autoimmune Diseases

• Cytokine-targeting therapies, such as mirikizumab, are being investigated for autoimmune diseases.
• B cell-targeting therapies, such as ianalumab, are being investigated for autoimmune diseases.
• Kinase-targeting therapies, such as evobrutinib and tolebrutinib, are being investigated for autoimmune diseases.

Potential Treatment for Autoimmune Diseases

• Cell therapies, such as CAR-T cells, CAR-Treg cells, and CAAR-T cells, are being investigated for autoimmune diseases.
• Targeting metabolic pathways, such as oxidative phosphorylation, mTOR, and glucose metabolism, is being investigated for autoimmune diseases.

Current and Potential Treatment for Systemic Lupus Erythematosus

• Rituximab, bortezomib, and atacicept are being investigated for SLE.
• CD19-targeting CAR-T cells are being investigated for refractory SLE.

Potential Target for Autoimmune Diseases

• Autoantibody-secreting plasma cells are a potential target for autoimmune diseases.
• Depleting plasma cells while redirecting the immune response towards non-autoreactive antigens is a potential strategy.

Future Perspectives for Treating Autoimmune Diseases

• New diagnostics, such as genome-wide screening for genes related to autoimmune diseases, are being developed.
• Personalized medicine, using high-throughput analysis of integrated datasets, is being developed.
• Preventive medicine, using polygenic risk scores and lifestyle modifications, is being developed.

Hallmarks of Cancer

• The hallmarks of cancer include sustained proliferative signaling, evading growth suppressors, and activating invasion and metastasis.
• Cancer cells acquire hallmark capabilities through genetic and epigenetic changes.

Cancer Development

• Cancer development is a multi-step process involving initiation, promotion, and progression.
• Genetic changes, such as mutations in oncogenes and tumor suppressor genes, drive cell transformation.

Invasive and Metastatic Cancer

• Cancer cells require critical traits to undergo invasion and metastasis.
• VEGF expression is increased in invasive and metastatic cancer.

Hallmarks of Cancer Cells

• Hallmarks of cancer cells include sustained proliferative signaling, evading growth suppressors, and activating invasion and metastasis.
• Cancer cells acquire hallmark capabilities through genetic and epigenetic changes.

Positive and Negative Regulators of Proliferation

• Positive regulators of proliferation, such as growth factors and integrin signaling, promote proliferation.
• Negative regulators of proliferation, such as E-cadherin, inhibit proliferation.

Hallmark 1: Sustained Proliferative Signaling

• Cancer cells acquire pro-proliferative signaling through genetic and epigenetic changes.
• Overexpression of growth factor receptors, such as HER2, renders cancer cells hyper-responsive to pro-proliferation signals.

Hallmark 2: Evading Growth Suppressors

• Cancer cells are resistant to anti-proliferative signals via E-cadherin loss.
• E-cadherin is a tumor suppressor gene that suppresses proliferation via cell-to-cell contact inhibition.