therapeutic strategies in genetic diseases

what is a genetic disorder?

• disease caused in whole or in part by a change in the DNA sequence away from the normal sequence

• dominant/recessive

• 3 categories: monogenetic, multifactorial inheritance, chromosome disorders

what are monogenetic disorders?

• caused by a mutation in a single gene

• e.g. sickle cell disease, cystic fibrosis, polycystic kidney disease, Tay-Sachs disease

• relatively rare

what are multifactorial inheritance disorders?

• caused by a combination of small inherited variations in genes, often acting together with environmental factors
• e.g. heart disease, diabetes, obesity, mental illness, Alzheimer's disease, most cancers

what are chromosomal disorders?

• caused by an excess/deficiency of the genes that are located on chromosomes or by structural changes within chromosomes
• e.g. down syndrome (trisomy 21), chronic myeloid leukemia (translocation in chr9 and chr22)

what is hemophilia A?

• X-linked recessive bleeding disorder that affects 1 in 5,000-10,000 males
• caused by mutations in the F8 gene (chr Xq28) that codes for coagulation factor VIII: essential cofactor in the coagulation pathway
• CDC hemophilia A mutation project (CHAMP) list: >2,500 mutations on F8 gene
• mutations reported to cause HA may be characterized by multiple mechanisms: deletion, duplication, insertion, inversion, substitution
• congenital deficiency in blood coagulation factor VIII results in excessive bleeding
• severe HA: <1% of the normal F8 protein level

what is the process of the human blood clotting cascade?

intrinsic pathway:
→ factors XII, XI, IX, VIII
→ activated partial thromboplastin time (aPTT)
extrinsic pathway:
→ tissue factor VII
→ prothrombin time (PT)

→ factor X
→ factor V: prothrombin
→ thrombin
→ fibrinogen
→ fibrin clot

what are the insufficient levels of factor FVII?

• the main medication to treat hemophilia A is concentrated FVIII product: plasma-derived or recombinant
• plasma derived FVIII has risks of hepatitis B and C and HIV
• several recombinant forms of factor VIII are available, but with a short half-life of 15-19 hrs

what is factor VIII?

• made by cells in the liver
• circulates in the bloodstream in an inactive form, bound to von willebrand factor (VWF), until an injury that damages a blood vessel occurs
• in response to injury, activated and separates from von willebrand factor
• active protein (FVIIIa) interacts with another coagulation factor IX: sets off a chain of additional chemical reactions that form a blood clot

what is the fusion protein for hemophilia A?

• BIVV001 is designed to uncouple recombinant factor VIII from von willebrand factor in circulation
• consists of a single recombinant factor VIII protein fused to dimeric Fc, a D'D3 domain of VWF, and 2 XTEN polypeptides
• fusion proteins has 4 times longer half-life

what are the components of the fusion protein for hemophilia A?

• factor VIII: at the core of ALTUVIIO is rFVIII, which replaces deficient FVIII in patients with hemophilia A
• Fc domain: delays FVIII degradation and helps FVIII stay in circulation by slowing the clearance rate
• XTEN polypeptide insertion: shields the FVIII molecule from proteolytic degradation and reduces binding to clearance receptors
• vWF D'D3 domain: prevents binding of FVIII to endogenous vWF

what is a better treatment for HA?

• valoctocogene roxaparvovec
• reduces bleeding episodes

what is cystic fibrosis?

• complex, chronic disease that primarily affects the lungs and digestive system, due to buildup of thick mucus in the lungs, pancreas, and other organs
• ~30,000 people in the US have CF
• autosomal recessive disease
• caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR)

what are the major functional deficits of CF?

misfolded and trapped in the endoplasmic reticulum, deficient in activation when reached in cell membrane

what is the CFTR?

• serves as a gated channel for Cl- ions, helping to maintain the balance of salt and water in the lungs, pancreas, GI, and sweat glands
• affected by chronic bacterial airway infection, neutrophilic inflammation, advanced CF lung disease

what types of mutations are there in the CFTR gene?

• >1,700 mutations
• nonsense/stop mutation: G542X, class I
• protein-processing mutation: F508del, class II, >80% of patients
• gating mutation: G551D, class III
• other mutations: R711H, class IV

what are class I CF mutations?

• readthrough small molecules
• mutated CFTR transcription → unstable mRNA

what are class II CF mutations?

• correctors + potentiator
• mutated CFTR transcription → unstable mRNA → misfolded CFTR → no Cl- trafficking

what are class III CF mutations?

• potentiator
• mutated CFTR transcription → unstable mRNA → misfolded CFTR → channel gate blocked

what are class IV CF mutations?

• potentiator
• mutated CFTR transcription → unstable mRNA → misfolded CFTR → faulty channel

what are class V CF mutations?

• potentiator
• mutated CFTR transcription → unstable mRNA → misfolded CFTR → not enough CFTR at cell surface

what are class VI CF mutations?

• potentiator
• mutated CFTR transcription → unstable mRNA → misfolded CFTR → high CFTR turnover

what is the combination therapy for CF?

defect 1:

• fewer CFTR proteins get to the cell surface, where they are normally located
• use correctors for Phe508del CFTR mutation
• by binding to different places on CFTR proteins, elexacaftor and tezacaftor work together to help more proteins reach the cell surface
  defect 2:
• CFTR proteins don't open correctly if they do reach the cell surface
• use potentiator for G551D gating mutation
• ivacaftor helps CFTR proteins stay open longer at the cell surface

what is the cause and therapy of CF?

• cause: loss of CFTR function
• therapy: delivery of CFTR mRNA packaged in lipid nanoparticles to drive expression of functional CFTR

what is duchenne muscular dystrophy (DMD)?

• recessive, X-linked (1 per 5,000 male births
• gene: dystrophin (DMD gene, chr Xp21, siez 2.4 Mb, 79 exons)
• mutation: large deletions caused by stop mutations, splicing mutations, deletions, and duplications
• phenotype: progressive degeneration of muscle mass, muscle-wasting disorder
• most commonly caused by out-of-frame intragenic deletions of one or more exons (60-70%), which produce premature transcript termination, leading to loss of the dystrophin protein

what is the therapy for DMD?

• exon skipping antisense oligomers to reframe transcripts, splicing modifiers, gene therapy
• AON hides exon 51 from splicing machinery: exon 51 is skipped and reading fram of transcript restored
• protein is internally deleted, but partially functional dystrophin

what is hutchinson-gilford progeria syndrome?

• rare autosomal dominant premature aging disorder
• accelerated cardiovascular disease from the buildup of defective progerin or progerin-like protein in cells
• most patients die before the age of 15 yrs from heart failure, heart attack, or stroke
• results from the buildup of progerin: mutant form of lamin A with a 50-amino acid deletion in the tail domain that leads to permanent farnesylation and membrane accumulation

what is persistent farnesylation?

• causes it to intercalate into the inner nuclear membrane, exerting damage to cells as they age
• the cellular instability appears to lead to the process of premature aging in progeria

what is the gene involved in hutchinson-gilford progeria syndrome?

• LMNA gene, chr 1q21.2: encodes the nuclear scaffold lamin A protein
• lamin A plays a critical role in nuclear envelope integrity and chromosome organization

what is the mutation that causes hutchinson-gilford progeria syndrome?

• mutation: a de novo single-base substitution within the LMNA gene exon 11 ( to T transition at nucleotide 1824)
• mutation activates a cryptic splice site and results in the production of a farnesylated mutant lamin A protein (progerin)
• leads to an in-frame deletion of 50 amino acids near the C-terminus of prelamin A
• splicesome cuts and deletes 50 amino acids

what are possible HGPS therapies?

• gene correction: Zn-fingers, TALENs, CRISPR
• pre-mRNA splicing correction: antisense approaches, small molecules
• RNA elimination: shRNA, RNAase H-mediated degradation
• progerin correction: FTIs, statins, biphosphonates
• progerin function: resveratrol
• progerin turnover: rapamycin
• reversal of cellular defects: epigenetic reprogramming, anti-inflammatories
• cell replacement therapies

what is antisense therapy for HCPS?

a targeted antisense therapeutic approach using antisense peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs)

what is ataxia telangiectasia?

• autosomal recessive mode of inheritance (1 per 40,000)
• gene: ATM (ataxia telangiectasia mutated, chr11q22.3)
• ATM is a key regulator of signaling following DNA double-strand breaks (DSBs)
• first sign: lack of balance, difficulty walking, and slurred speech, later use a walker or wheelchair
• signs: spider veins (telangiectases, dilated blood vessels)

what are the mutations in the ATM gene?

• reduce/eliminate the function of the ATM protein
  leads to:
• polyploidy
• chromosomal abnormalities and breaks
• DNA repair defects
• failure to arrest DNA synthesis or mitosis following exposure to ionizing radiation

what is xeroderma pigmentosum (XP)?

• autosomal recessive genetic disease
• xeroderma (dry, scaly sking) and pigmentosum (freckling and abnormal skin coloring) refer to changes that occur after exposure to sunlight or other UV radiation
• people with this condition develop skin and eye cancers at young ages because their DNA is extremely susceptible to damage caused by UV radiation
• those lack the enzymatic ability to recognize and excise thymine-thymine dimers produced by UV light

what are the signs of XP?

• 9 months: severe blistering
• 2 yrs: squamous cell carcinoma
• 23 yrs: nodular basal cell cancer, cornea scar
• 35 yrs: neurological degeneration, hyperpigmentation, deafness

what is the DNA repair and therapy for XP?

• part of nucleotide excision repair (NER)
• inability to correct damaged DNA by sunlight results in mutations
• standard therapy: no therapy available; prevention and dermatologic care

what are the genes associated with XP?

XPA (chr9), ERCC3 (XPB, chr2), XP3 (XPC, chr3O, ERCC2 (XPD, chr19), DDB2 (XPE, chr11), ERCC4 (XPF, chr16), ERCC4 (XPG, chr13), POLH (XPV, chr6)

what is turner's sundrome?

• abnormal # of sex chromosomes
• genetic defect: 45X
• frequency: 1.3
• features: female gender, broad chest, undeeloped ovaries

what is klinefelter's syndrome?

• abnormal # of sex chromosomes
• genetic defect: 47XXY
• frequency: 0.1
• features: male gnder with female habit

what is down's syndrome?

• abnormal # of autosomes
• genetic defect: 47, trisomy 21 (95% cases)
• frequency: 1.4
• features: wide skull, upward slanting eyes, flat nasal bridge, mental subnormality, congenital heart defects

what is patau's syndrome?

• abnormal # of autosomes
• genetic defect: 47, trisomy 13
• frequency: 0.1
• features: microcephaly, small eyes, cleft palate, low-set ears

what is hereditary cancer?

only ~5-10% of all cancers result directly from gene defects inherited from a parent

what are the types of hereditary cancer?

• breast and ovarian: BRCA1/BRCA2 gene
• li-fraumeni syndrome (sarcoma, leukemia, breast cancer): TP53 gene
• lunch syndrome (hereditary non-polyposis colorectal cancer): several mismatch repair (MMR) genes, including MLH1, MSH2, MSH6, PMS1, PMS2

what is cancer risk?

• the lifetime cancer risk is correlated with the division rate of the cell of origin of the cancer
• cancer-causing genetic changes accumulate slowly as a person ages

what are the general steps of drug development in the era of precision medicine?

1. indication
2. biomarker
3. technology
4. drugs

what are the steps of precision medicine and companion diagnostics of HER2?

1. indication: breast cancer
2. biomarker: HER2 (ERBB2) amplification
3. technology: HercepTest (semi-quantitative immunohistochemical assay), FISH, CISH
4. drug: trastuzumab (herceptin)

what are the steps of precision medicine and companion diagnostics I of EGFR?

1. indication: non-small-cell lung cancer
2. biomarker: EGFR mutations (e.g. exon 19 deletion, exon 21 (L858R) substitution mutations)
3. technology: real-time PCR
4. drug: afatinib (gilotrif)

what are the steps of precision medicine and companion diagnostics II of EGFR?

1. indication: breast cancer
2. biomarker: EGFR^T790M mutations
3. technology: real-time PCR
4. drug: osimertinib (tagrisso)

what are the steps of precision medicine and companion diagnostics of BRCA?

1. indication: ovarian cancer
2. biomarker: BRCA1/BRCA2 mutations
3. technology: PCR and sanger sequencing
4. drug: plaparib (lynparza)

what are the steps of precision medicine and companion diagnostics of philadelphia chromosome?

1. indication: chronic myelogenous leukemia (CML)
2. biomarker: philadelphia chromosome
3. technology: karyotyping and FISH, required for diagnosis of CML
4. drug: gleevec (imantinib)

what is gleevec (imatinib)?

• tyrosine kinase inhibitor
• rationally designed to target the abl protein (tyrosine kinase) and prevent it from transmitting the signal that triggers the cell to divide

what is CML?

• caused by excess production of WBCs
• produces an abnormal gene called bcr-abl which results in the abl protein continuing to signal and the WBCs to keep dividing