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#Factor VIII

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Gene Therapy

Factor VIII recombinant protein is commonly produced in:

Coagulation factor VIII complex multidomain glycoprotein organized signal peptide 19 residues followed domains A1 a1 A2 a2 B a3 A3 C1 C2 totaling 2351 aa 280 kDa requiring extensive co-translational post-translational processing secretory pathway. Nascent polypeptide translocates Sec61 translocon ER lumen signal peptidase cleaves signal oligosaccharyltransferase adds high-mannose oligosaccharides 25 asparagine X Ser Thr motifs protein disulfide isomerase forms 8 disulfide bonds calnexin calreticulin cycle glucosidase II monitors folding peptidyl prolyl isomerase assists. Golgi further trims mannose adds complex sialylated glycans glycosyltransferases sulfates tyrosines 346 718 719 tyrosylprotein sulfotransferase essential vWF binding incorporates copper ions. E. coli cytoplasm lacks ER glycosylation sulfation chaperone BiP leading misfolded aggregates inclusion bodies lacking cofactor activity eliciting neutralizing antibodies exposed neoepitopes. Chinese hamster ovary cells possess mammalian processing enzymes secrete active factor serum-free medium supplemented vWF stabilizing. Stable clones expressing B-domain-deleted FVIII CMV promoter purified monoclonal antibody immunoaffinity anion exchange preserving specific activity around 5000 IU per mg therapeutic use.

Ref: Blood Factor VIII CHO Production Kaufman 1988; FDA Recombinant Factor VIII Manufacturing Guidelines; Alberts Protein Glycosylation Mammalian Cells Chap 15.

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Gene Therapy

Hemophilia gene therapy targets deficiency of:

Hemophilia A 1 in 5000 males and B 1 in 30000 males result null mutations coagulation cofactors FVIII 280 kDa glycoprotein encoded F8 Xq28 domains A1 A2 B A3 C1 C2 and FIX vitamin K dependent serine protease encoded F9 Xq27 gamma carboxyglutamic acid residues binding calcium phospholipid. Both assemble intrinsic tenase complex FVIIIa cofactor enhancing FIXa protease activity 200000-fold toward FX activation on phosphatidylserine-rich platelet surface accelerating thrombin burst fibrin clot formation. Deficiency prolongs aPTT causing hemarthrosis, muscle hematoma. Replacement requires frequent IV infusions half-life FVIII 12h FIX 18h. Gene therapy delivers codon-optimized B-domain-deleted F8 central 908 aa dispensable removed reducing size 4.4 kb fitting AAV, or FIX Padua variant R338L leucine substitution arginine 338 increasing specific activity 8-fold improving secretion. Hepatotropic AAV5 AAV6 liver-specific promoter HLP transcribe hepatocytes synthesize factor secreting into sinusoids restoring plasma levels 5-150 percent converting severe to mild reducing annualized bleeding rate 90 percent in trials Hemgenix Roctavian leading to regulatory approvals.

Ref: NEJM Hemophilia Gene Therapy Nathwani 2022; FDA Hemgenix Roctavian Approvals; Alberts Cell Biology Coagulation Cascade Chap 20.

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