L09 Genetics Recombinant proteins, gene expression, factor 8

Overview

This lecture uses Haemophilia A as a worked example of a monogenic disease, covering three things: the genetics of the Factor VIII gene and how its mutations cause disease of varying severity, how the Factor VIII protein is built and activated and how mutations disrupt it, and how the clotting factor used to treat the disease has been sourced over time, from pooled plasma through recombinant protein production to antibody-based and gene-therapy alternatives.

Haemophilia A: presentation and basic management

• Symptoms: intracranial haemorrhage, prolonged nosebleeds, easy bruising, warm/painful/swollen joints with reduced movement, and GI haemorrhage, which presents as coffee-ground emesis, cola-coloured urine and tarry stools.
• General management (no cure exists): avoid injury and medications that promote bleeding, maintain good nutrition and good dental hygiene, and give the deficient clotting factor intravenously.
• Factor VIII is a cofactor that helps factor IXa activate factor X to Xa in the coagulation cascade.
• Clot inhibitor drugs: warfarin inhibits the vitamin K needed for hepatic synthesis of clotting factors; dabigatran directly blocks thrombin activity.
• Most Haemophilia A is genetic, but rare cases are acquired (autoimmune, antibodies form against factor VIII). Acquired cases are treated differently, bypassing the need for factor IX with recombinant factor VII.
• Severity is classed by residual factor activity: severe <1%, moderate 1-5%, mild 5-30%.

Inheritance and pedigree interpretation

• X-linked recessive. Before treatment existed it was effectively X-linked lethal. Incidence is about 1 in 5000 males.
• Some carrier females (about 10%) are clinically affected, through skewed X-inactivation: enough cells preferentially inactivate the X carrying the normal allele that factor VIII activity is low.
• Worked pedigree logic: an unaffected father (XHY) and carrier mother (XHXh) can have sons who are XHY (unaffected) or XhY (affected), and daughters who are XHXH (unaffected) or XHXh (carrier).

The FVIII gene and its mutations

• Location and size: Xq28, near the telomere; about 186 kb; 26 exons; 9 kb cDNA.
• Over 600 mutations have been described (allelic heterogeneity), which complicates molecular analysis. Some polymorphisms are normal variants that do not cause disease.
• Mild-moderate disease: about 90% of cases are point mutations or small deletions, about 10% are other changes such as splice-site mutations or rare promoter mutations; residual enzyme activity is 1-30%.

Severe disease is dominated by one recurrent structural mutation, not point mutations: about 40% of severe cases are caused by a specific intron-22 inversion, leaving under 1% residual protein activity.

• Mechanism of the intron-22 inversion: the gene locus carries an intragenic inverted repeat (Int22h-1, near exon 22), a proximal inverted repeat (Int22h-2, about 0.3 Mb away) and a distal inverted repeat (Int22h-3, a further 0.1 Mb away), oriented inverted relative to one another. The distal repeat loops back and pairs with the intragenic repeat by homologous recombination. The result is that the segment spanning exons 1-22 is inverted while exons 23-26 remain in place, disrupting the gene and producing a truncated protein. This event is more likely in male meiosis because there is no homologous X chromosome available to pair with instead.

From gene to protein, and how mutations disrupt it

• FVIII mRNA is 9010 nt total: 5’-UTR 150 nt, coding sequence plus stop codon 7056 nt, 3’-UTR 1806 nt.
• The inactive Factor VIII protein has a signal peptide followed by domains A1-A2-B-A3-C1-C2 (with short linking “a” segments released on activation). Thrombin cleaves it at arginines 372, 740 and 1689 to produce active Factor VIIIa: a heavy chain (A1-A2) joined via Ca2+ to a light chain (A3-C1-C2). Further cleavage sites elsewhere in the protein are acted on by other proteases (factor Xa, activated protein C, factor IXa), some intracellular and some extracellular.
• Factor VIII is synthesised in the liver and processed through the endoplasmic reticulum; multiple glycosylations (a post-translational modification) are required for it to function, which is why it must be produced in mammalian cells.
• Regions of the folded protein mediate von Willebrand factor (vWF) binding and phospholipid (PL) surface binding.
• A mutation can disrupt Factor VIII function by: changing the thrombin cleavage sites and preventing activation, changing binding to vWF, altering secretion, altering interaction with factor IX or X, or destabilising protein folding.

Treatment history: plasma and plasma-derived concentrates

• Three product eras have been used to increase survival and reduce complications: plasma (historical, required hospitalisation), Factor VIII concentrates purified from plasma (from the 1960s), and recombinant Factor VIII (from 1992 onward). NZ currently has about 350 Haemophilia A patients and 90 Haemophilia B patients.
• Plasma-derived concentrate aims to raise factor levels to 30-50% during a bleed. It is purified in a complex with vWF, concentrated from large pools of donated blood by biochemical techniques, and raised life expectancy to near normal. From the 1980s onward there were growing concerns about blood safety.
• Heat treatment used in processing may not fully inactivate non-enveloped viruses such as parvovirus B19 and Torque Teno Virus (TTV); enveloped viruses such as HIV are removed, but prions are not removed or inactivated by these methods (illustrated by the rapid brain shrinkage and spongiform pathology of Creutzfeldt-Jakob disease).
• Biostate is a plasma-derived concentrate made in Australia from NZ donors, containing both Factor VIII and vWF. It remains in clinical use in NZ for patients who have been established on it for years.

Recombinant Factor VIII: production and current NZ products

• Recombinant Factor VIII is produced in CHO (Chinese hamster ovary) or BHK (baby hamster kidney) cells rather than bacteria, because these mammalian lines grow well in culture (about 40 years of experience), are well studied, had prior use for recombinant proteins, resist infection, are free of human pathogens, and can perform the glycosylation Factor VIII needs for a normal plasma half-life.
• Production process: FVIII cDNA is cloned into an rDNA vector carrying a mammalian promoter and a mammalian polyadenylation signal; the construct is introduced into mammalian cells, which ensures correct post-translational modification; cells are selected for the transfer marker and for transgene expression; selected cells are grown in a bioreactor, in media containing fetal calf serum and nutrients or in defined serum-free media with added human protein mixes.
• Purification from the bioreactor product takes many steps (about 13), which may include immuno-affinity purification with a specific antibody. The steps, in order: ion exchange chromatography, heat and chemical treatment, immuno-affinity chromatography, gel permeation chromatography, a second immuno-affinity chromatography, a second ion exchange chromatography, DEAE chromatography, ultrafiltration, stabilisation with human albumin, a second heat and chemical treatment, diafiltration, and sterile filtration.
• Limitations: expression levels are still low and the tissue-culture and purification process is expensive; the glycosylation pattern differs slightly from the native protein but the product remains functional in humans.
• Current NZ-funded recombinant products (funded for prophylaxis in all patients since May 2019), neither containing vWF:
  • Advate: full-length Factor VIII including the B domain, cleaved for activation by thrombin, stabilised with polysorbate; short-acting, half-life 13-18 h.
  • Adynovate: full-length Factor VIII including the B domain, cleaved for activation by thrombin, stabilised with PEG (polyethylene glycol); long-acting, half-life 20-30 h.

The source pricing table lists two vial sizes ( $210.00for250IU,$420.00 for 500 IU) both labelled "Advate," even though the second row sits under/beside the Adynovate bullet text on the slide. This may be a source labelling error and is transcribed here exactly as shown, not corrected.

Factor VIII inhibitors and antibody-based alternative treatment

• Inhibitors are antibodies raised by the immune system against the infused (foreign) Factor VIII protein. They affect 5-7% of all patients and up to 30% of patients with severe Haemophilia A, at a similar rate whether plasma-derived or recombinant product is used. They are managed by immune tolerance induction (low, repeated doses), but treatment for inhibitors is difficult and expensive.
• Emicizumab (branded Hemlibra) is used when Factor VIII inhibitors are present. It is a bispecific antibody that binds both factor IXa and factor X, bridging them together on a phospholipid membrane to generate factor Xa in the same way that factor VIIIa normally does, so it mimics Factor VIII’s action rather than replacing it, avoiding the need to give Factor VIII at all.
• Emicizumab is a human-mouse-rat chimeric antibody. It was produced by raising separate mouse and rat monoclonal antibodies against factor X and factor IXa respectively, then engineering a vector that combines their variable-region sequences with human IgG4/kappa constant regions, with mutations enabling heavy-chain hetero-dimerisation, to assemble a four-chain bispecific IgG with a shared light chain.
• PHARMAC NZ funded emicizumab from 11 November 2020 for patients with severe Haemophilia A and Factor VIII inhibitors. Cost is about $400,000 per patient per year, and about 15 patients are on it in NZ.

Gene therapy and future directions

• The FDA approved (June 2023) a gene therapy for clinical use in adults with severe Haemophilia A. Patients must test negative for pre-existing AAV5 antibodies, which reduce the treatment’s effectiveness. Its list price is $2.9millionUSD,comparedwithanestimated$4.5 million USD lifetime cost of traditional Factor VIII therapy. As of the lecture, NZ had not considered funding it.
• Gene therapy more broadly: 42 FDA-approved gene therapy products are in clinical use and over 500 are in clinical trials. Approved products include genetically modified cells for ex vivo treatment, such as CAR-T cells (targeting patient-specific tumour antigens), CRISPR-based gene editing therapy, and a gene therapy for metachromatic leukodystrophy (a rare childhood disease).
• Expected future developments: better delivery vectors designed to target specific cell types and tissues, since targeting the wrong tissue (off-target delivery) can cause serious side effects including severe immune responses to the vector or transgene; methods to regulate gene expression, using optimised promoters that better mimic endogenous expression; and further optimisation of CRISPR to allow editing of mutations within endogenous genes.

Self-test

1. Describe the classic symptoms of Haemophilia A, including the three specific signs of GI haemorrhage.
2. Outline the general (non-factor-specific) management principles for Haemophilia A.
3. Describe Factor VIII’s role in the coagulation cascade, and explain how warfarin and dabigatran each inhibit clotting.
4. Distinguish genetic from acquired Haemophilia A, and explain why acquired cases are treated with recombinant factor VII rather than factor VIII.
5. Define the three severity classes of Haemophilia A by residual factor activity.
6. Describe the inheritance pattern and incidence of Haemophilia A.
7. Explain how a female carrier of Haemophilia A can become clinically affected.
8. Using the pedigree logic given (unaffected father, carrier mother), predict the possible genotypes of their sons and daughters.
9. State the chromosomal location and size of the FVIII gene (location, gene size, exon number, cDNA length).
10. Explain why the FVIII gene’s mutation spectrum complicates molecular diagnosis.
11. Describe the two major categories of FVIII mutation causing mild-moderate disease and their relative frequency.
12. Describe the steps of the intron-22 inversion mechanism that causes severe Haemophilia A, and explain why it occurs more often in male meiosis.
13. What proportion of severe Haemophilia A cases arise from the intron-22 inversion, and how much residual protein activity remains?
14. Describe the structure of FVIII mRNA, giving the length of each region.
15. Describe how thrombin activates Factor VIII, including the resulting protein structure.
16. List the ways a Factor VIII mutation can alter protein function.
17. Where is Factor VIII synthesised, and what post-translational modification is essential for its function?
18. Describe the three historical eras of Haemophilia A treatment products, with approximate dates.
19. Explain the main historical safety concern with plasma-derived Factor VIII concentrates, naming two relevant pathogens and why they posed different risks.
20. What is Biostate, and why does it remain in clinical use in NZ?
21. Explain why CHO or BHK mammalian cells, rather than bacteria, are used to produce recombinant Factor VIII.
22. Describe the process of producing recombinant Factor VIII, from gene construct to purified product.
23. Compare Advate and Adynovate in terms of composition, stabilisation chemistry and half-life.
24. Describe Factor VIII inhibitors: their cause, prevalence, and management.
25. Explain how emicizumab substitutes for Factor VIII activity, and describe the biological origin of its structure.
26. Describe the FDA-approved gene therapy for severe Haemophilia A: the patient exclusion criterion, and how its cost compares with lifetime traditional treatment.
27. Integrative: for a patient with severe Haemophilia A due to the intron-22 inversion who has developed Factor VIII inhibitors, explain why plasma-derived and recombinant Factor VIII infusions become ineffective, and why emicizumab remains a viable option.

Answers

Reveal answers

1. Intracranial haemorrhage, prolonged nosebleeds, easy bruising, warm/painful/swollen joints with reduced movement, and GI haemorrhage; the GI-bleed signs are coffee-ground emesis, cola-coloured urine and tarry stools.
2. No cure exists; avoid injury and medications that promote bleeding; maintain good nutrition; maintain good dental hygiene; give the deficient clotting factor intravenously.
3. Factor VIII is a cofactor that helps factor IXa activate factor X to Xa. Warfarin inhibits the vitamin K needed for hepatic synthesis of clotting factors; dabigatran directly blocks thrombin activity.
4. Genetic Haemophilia A is inherited (X-linked); acquired Haemophilia A is autoimmune, with antibodies forming against factor VIII. Acquired cases are treated by bypassing the need for factor IX with recombinant factor VII, since infused factor VIII would be neutralised by the antibodies.
5. Severe: <1% normal activity. Moderate: 1-5%. Mild: 5-30%.
6. X-linked recessive; before treatment existed it was effectively X-linked lethal; incidence is about 1 in 5000 males.
7. Through skewed X-inactivation: enough of her cells preferentially inactivate the X chromosome carrying the normal allele that her factor VIII activity is low enough to cause symptoms (occurs in about 10% of carriers).
8. Sons: XHY (unaffected) or XhY (affected). Daughters: XHXH (unaffected) or XHXh (carrier).
9. Xq28, near the telomere; about 186 kb; 26 exons; 9 kb cDNA.
10. Over 600 different mutations have been described (allelic heterogeneity), so a large number of possible causal variants must be checked, complicating molecular analysis.
11. About 90% are point mutations or small deletions; about 10% are other changes such as splice-site mutations or rare promoter mutations. These leave 1-30% normal enzyme activity.
12. Intragenic (Int22h-1), proximal (Int22h-2, about 0.3 Mb away) and distal (Int22h-3, a further 0.1 Mb away) inverted repeats flank the gene. The distal repeat loops back and pairs with the intragenic repeat by homologous recombination, inverting the segment containing exons 1-22 while exons 23-26 stay in place, disrupting the gene and producing a truncated protein. It occurs more often in male meiosis because there is no homologous X chromosome available to pair with instead.
13. About 40% of severe cases; residual activity is under 1%.
14. Total 9010 nt: 5’-UTR 150 nt, coding sequence plus stop codon 7056 nt, 3’-UTR 1806 nt.
15. Thrombin cleaves the inactive protein at arginines 372, 740 and 1689 to give active Factor VIIIa: a heavy chain (A1-A2) joined via Ca2+ to a light chain (A3-C1-C2).
16. Changing the thrombin cleavage sites and preventing activation; changing binding to von Willebrand factor; altering secretion; altering interaction with factor IX or X; destabilising protein folding.
17. Synthesised in the liver and processed through the endoplasmic reticulum; multiple glycosylations are required for it to function.
18. Plasma (historical, required hospitalisation); Factor VIII concentrates purified from plasma (from the 1960s); recombinant Factor VIII (from 1992 onward).
19. Heat treatment used to inactivate pathogens may not fully inactivate non-enveloped viruses such as parvovirus B19 and Torque Teno Virus, whereas enveloped viruses such as HIV are removed; prions (as in Creutzfeldt-Jakob disease) are not removed or inactivated by these methods at all.
20. A plasma-derived concentrate made in Australia from NZ donors, containing both Factor VIII and von Willebrand factor; it remains in use for NZ patients who have been established on it for years.
21. They grow well in culture (about 40 years of experience), are well studied, have prior recombinant-protein production history, resist infection, are free of human pathogens, and can perform the glycosylation Factor VIII needs for a normal plasma half-life, which bacteria cannot do.
22. FVIII cDNA is cloned into an rDNA vector with a mammalian promoter and polyadenylation signal; the construct is introduced into mammalian cells (e.g. CHO), ensuring correct post-translational modification; cells are selected for the transfer marker and transgene expression; selected cells are grown in a bioreactor; the protein is then purified from the bioreactor product over about 13 steps, which may include immuno-affinity purification with a specific antibody.
23. Both are full-length Factor VIII including the B domain, cleaved for activation by thrombin, and neither contains von Willebrand factor. Advate is stabilised with polysorbate and is short-acting (half-life 13-18 h); Adynovate is stabilised with PEG and is long-acting (half-life 20-30 h).
24. Antibodies raised by the immune system against infused Factor VIII; they affect 5-7% of all patients and up to 30% of patients with severe Haemophilia A, at a similar rate for plasma-derived and recombinant products; managed by immune tolerance induction using low, repeated doses, though treatment is difficult and expensive.
25. Emicizumab binds both factor IXa and factor X, bridging them on a phospholipid membrane to generate factor Xa in the same way factor VIIIa normally does, mimicking rather than replacing Factor VIII. It is a human-mouse-rat chimeric antibody, built by raising mouse and rat monoclonal antibodies against factor X and factor IXa, then engineering a vector combining their variable regions with human IgG4/kappa constant regions to produce a four-chain bispecific IgG with a shared light chain.
26. A gene therapy FDA-approved in June 2023 for adults with severe Haemophilia A; patients must be negative for pre-existing AAV5 antibodies, which reduce effectiveness. Its list price is $2.9millionUSD,versusanestimated$4.5 million USD lifetime cost of traditional Factor VIII therapy.
27. The intron-22 inversion produces a severely truncated Factor VIII protein, so infused plasma-derived or recombinant Factor VIII is recognised as foreign and drives inhibitor (antibody) formation; once inhibitors are present, infused Factor VIII of either source is neutralised before it can act. Emicizumab remains effective because it is a bispecific antibody structurally unrelated to Factor VIII that bridges factor IXa and factor X directly, so it is not blocked by anti-Factor VIII inhibitors.