<i>In vivo</i> efficacy of human recombinant factor IX produced by the human hepatoma cell line HuH‐7

Enjolras, Nathalie; Perot, Eloïse; Quellec, Sandra Le; Indalecio, Alice; Girard, J.; Négrier, Claude; Dargaud, Yesim

doi:10.1111/hae.12688

Cited by 4 publications

(2 citation statements)

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“…CHO cells were shown to produce active FIX in-vitro to levels almost comparable with primary hepatocytes and they are harnessed to purify FIX for injection into hemophilic patients [22]. And indeed, transfection of the minigenes into CHO cells could reproduce the phenotype at the level of RNA ( Fig 3A) and intracellular FIX protein ( Fig 3B, lanes 4,5 and 6,7).…”

Section: The Improved Fix Minigene Phenocopies the Mutant Also In Redmentioning

confidence: 93%

Pathological mechanism and antisense oligonucleotide-mediated rescue of a non-coding variant suppressing factor 9 RNA biogenesis leading to hemophilia B

et al. 2020

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Loss-of-function mutations in the human coagulation factor 9 (F9) gene lead to hemophilia B. Here, we dissected the consequences and the pathomechanism of a non-coding mutation (c.2545A>G) in the F9 3' untranslated region. Using wild type and mutant factor IX (FIX) minigenes we revealed that the mutation leads to reduced F9 mRNA and FIX protein levels and to lower coagulation activity of cell culture supernatants. The phenotype could not be compensated by increased transcription. The pathomechanism comprises the de novo creation of a binding site for the spliceosomal component U1snRNP, which is able to suppress the nearby F9 poly(A) site. This second, splicing-independent function of U1snRNP was discovered previously and blockade of U1snRNP restored mutant F9 mRNA expression. In addition, we explored the vice versa approach and masked the mutation by antisense oligonucleotides resulting in significantly increased F9 mRNA expression and coagulation activity. This treatment may transform the moderate/severe hemophilia B into a mild or subclinical form in the patients. This antisense based strategy is applicable to other mutations in untranslated regions creating deleterious binding sites for cellular proteins.

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Section: The Improved Fix Minigene Phenocopies the Mutant Also In Redmentioning

confidence: 93%

Pathological mechanism and antisense oligonucleotide-mediated rescue of a non-coding variant suppressing factor 9 RNA biogenesis leading to hemophilia B

et al. 2020

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“…Huh‐7 cells (ECACC 01042712) were maintained in conditioned media containing 5 µg/mL of vitamin K as described previously and were transiently transfected with 1 µg of pAV‐LP1‐hFIXco‐Alb, pAV‐LP1‐hFIXco‐STOP‐(Alb), pAV‐LP1‐hFIXco‐Fc and pAV‐LP1‐hFIXco‐STOP‐(Fc) vectors using Fugene ® 6 (Roche, Neuilly‐sur‐Seine, France). Huh‐7 cells were also transduced in vitro at an MOI of 10 5 vg/cell of all four corresponding ssAAV2/8 vectors, as previously reported .…”

Section: Methodsmentioning

confidence: 99%

Potential limits of AAV‐based gene therapy with the use of new transgenes expressing factor IX fusion proteins

et al. 2018

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Introduction The variety of treatment for haemophilia B (HB) has recently improved with the emergence of both AAV‐based gene therapy and bioengineered human factor IX (hFIX) molecules with prolonged half‐life due to fusion to either albumin (Alb) or immunoglobulin Fc fragment (Fc). Aim Adeno‐associated viral vectors (AAV) mediating expression of hFIX‐Alb and hFIX‐Fc fusion proteins was investigated for gene therapy of HB to explore if their extended half‐life translates to higher plasma levels of FIX. Methods Single‐stranded cross‐packaged AAV2/8 vectors expressing hFIX‐Alb, hFIX‐Fc and hFIX were evaluated in vitro, and in mice. Results Both hFIX‐Alb and hFIX‐Fc fusion proteins were synthesized and expressed as single chains of expected size following AAV‐mediated gene transfer in vitro and in vivo. The procoagulant properties of these hFIX‐fusion proteins were comparable to wild‐type hFIX. However, their expression levels were threefold lower than wild‐type hFIX in vivo most likely due to inefficient secretion. Conclusion This, the first, evaluation of hFIX‐fusion proteins in the context of AAV gene transfer suggests that the hFIX‐fusion proteins are secreted inefficiently from the liver, thus preventing their optimal use in gene therapy approaches.

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Lindskog¹,

Fischer²,

Wenger³

et al. 2018

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In vivo efficacy of human recombinant factor IX produced by the human hepatoma cell line HuH‐7

Abstract: The study shows that HuH-7-derived-rFIX has better in vivo haemostatic activity in hemophilia B mice compared to the reference rFIX molecule despite similar in vivo recovery rates, suggesting that HuH-7 cells could represent an effective cellular system for production of rFIX.

Cited by 4 publications

References 18 publications

Pathological mechanism and antisense oligonucleotide-mediated rescue of a non-coding variant suppressing factor 9 RNA biogenesis leading to hemophilia B

Pathological mechanism and antisense oligonucleotide-mediated rescue of a non-coding variant suppressing factor 9 RNA biogenesis leading to hemophilia B

Potential limits of AAV‐based gene therapy with the use of new transgenes expressing factor IX fusion proteins

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