X-Linked Thrombophilia with a Mutant Factor IX (Factor IX Padua)

Simioni, Paolo; Tormene, Daniela; Tognin, Giulio; Gavasso, Sabrina; Bulato, Cristiana; Iacobelli, Nicholas P.; Finn, Jonathan D.; Spiezia, Luca; Radu, Claudia; Arruda, Valder R.

doi:10.1056/nejmoa0904377

Cited by 303 publications

(271 citation statements)

References 21 publications

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“…We then combined these hepatocyte-specific CRMs (HS-CRMs) with a synthetic codon-optimized hyperfunctional FIX transgene (ie, Padua R338L) that conferred 15-fold higher expression and activity levels than its wild-type counterpart. 6,7 This novel combination approach substantially reduced the dose requirement for reaching therapeutic efficacy and thus facilitates future scale-up and clinical translation. There is an Inside Blood Commentary on this article in this issue.…”

Section: Introductionmentioning

confidence: 99%

Computationally designed liver-specific transcriptional modules and hyperactive factor IX improve hepatic gene therapy

Nair¹,

Rincón

Evens³

et al. 2014

Blood

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Key Points• Liver-targeted gene therapy for hemophilia can be improved by using computational promoter design in conjunction with hyperfunctional FIX.• Low and safe vector doses allow for stable supraphysiologic FIX that result in the induction of immune tolerance.The development of the next-generation gene therapy vectors for hemophilia requires using lower and thus potentially safer vector doses and augmenting their therapeutic efficacy. We have identified hepatocyte-specific transcriptional cis-regulatory modules (CRMs) by using a computational strategy that increased factor IX (FIX) levels 11-to 15-fold. Vector efficacy could be enhanced by combining these hepatocyte-specific CRMs with a synthetic codon-optimized hyperfunctional FIX-R338L Padua transgene. This Padua mutation boosted FIX activity up to sevenfold, with no apparent increase in thrombotic risk. We then validated this combination approach using self-complementary adenoassociated virus serotype 9 (scAAV9) vectors in hemophilia B mice. IntroductionSignificant progress has recently been made toward the development of gene therapy for hemophilia B. Adenoassociated virus (AAV) vectors are among the most promising vectors for liver-directed gene therapy that are capable of achieving therapeutic factor IX (FIX) expression levels in patients suffering from severe hemophilia B. 1,2 Nevertheless, there are still some issues related to the induction of AAV capsidspecific T-cell-mediated immune response against the AAV-transduced cells that need to be addressed. [1][2][3][4] These inadvertent immune reactions curtailed long-term gene expression by eliminating the gene-modified cells and accounted for liver toxicity. Furthermore, the performance of these AAV vectors must be improved to achieve a bona fide cure. Consequently, there is a need to create the next-generation AAV vectors for liver-directed gene therapy that express higher FIX levels at lower vector doses, to the extent that stable physiologic levels of FIX can be attained, while preventing inadvertent AAV capsid-specific T-cell responses and liver toxicity. The availability of more potent vectors would also ease manufacturing needs. To increase the potency of AAV-FIX vectors, we explored the use of a bioinformatics algorithm that resulted in the identification of transcriptional cis-regulatory modules ( 5 These CRMs contained evolutionary conserved clusters of transcription factor binding-site motifs that confer high tissue-specific gene expression. We then combined these hepatocyte-specific CRMs (HS-CRMs) with a synthetic codon-optimized hyperfunctional FIX transgene (ie, Padua R338L) that conferred 15-fold higher expression and activity levels than its wild-type counterpart.6,7 This novel combination approach substantially reduced the dose requirement for reaching therapeutic efficacy and thus facilitates future scale-up and clinical translation. There is an Inside Blood Commentary on this article in this issue.The publication costs of this article were defrayed in part by page charge payment. T...

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Section: Introductionmentioning

confidence: 99%

Computationally designed liver-specific transcriptional modules and hyperactive factor IX improve hepatic gene therapy

Nair¹,

Rincón

Evens³

et al. 2014

Blood

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“…16 Here, in the AAV-cFIXPadua-expressing dogs, there was no detection of antibodies to cFIX by Bethesda assay or anti-cFIX IgG2 ELISA ( Figure 1B,E and supplemental Figure 2). Thus, expression of the cFIX-Padua variant shows increased specific activity as observed in humans 12 and immune responses even on challenge with cFIX-WT protein concentrates.…”

Section: Canine Studiesmentioning

confidence: 83%

“…The mutant FIX circulates at normal antigenic levels but exhibits eightfold increased clotting activity. 12 Thus, the use of FIX-Padua offers an alternative strategy for treating HB. Preclinical studies in severe HB dogs provide a unique opportunity to address both efficacy and safety.…”

Section: Introductionmentioning

confidence: 99%

AAV liver expression of FIX-Padua prevents and eradicates FIX inhibitor without increasing thrombogenicity in hemophilia B dogs and mice

Crudele

Finn

Siner

et al. 2015

Blood

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137

175

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• Liver-restricted expression of FIX-Padua induces immune tolerance to the transgene in hemophilia B inhibitor dog models.• Long-term toxicity studies show no increased risk of thrombogenicity of FIX-Padua in mice and dogs.Emerging successful clinical data on gene therapy using adeno-associated viral (AAV) vector for hemophilia B (HB) showed that the risk of cellular immune response to vector capsid is clearly dose dependent. To decrease the vector dose, we explored AAV-8 (1-3 3 10 12 vg/kg) encoding a hyperfunctional factor IX (FIX-Padua, arginine 338 to leucine)in FIX inhibitor-prone HB dogs. Two naïve HB dogs showed sustained expression of FIX-Padua with an 8-to 12-fold increased specific activity reaching 25% to 40% activity without antibody formation to FIX. A third dog with preexisting FIX inhibitors exhibited a transient anamnestic response (5 Bethesda units) at 2 weeks after vector delivery following by spontaneous eradication of the antibody to FIX by day 70. In this dog, sustained FIX expression reached ∼200% and 30% of activity and antigen levels, respectively. Immune tolerance was confirmed in all dogs after challenges with plasma-derived FIX concentrate. Shortening of the clotting times and lack of bleeding episodes support the phenotypic correction of the severe phenotype, with no clinical or laboratory evidence of risk of thrombosis. Provocative studies in mice showed that FIX-Padua exhibits similar immunogenicity and thrombogenicity compared with FIX wild type. Collectively, these data support the potential translation of gene-based strategies using FIX-Padua for HB. (Blood. 2015;125(10):1553-1561

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“…34 Recently, the importance of this particular residue was highlighted by the identification of a family with a potent X-linked thrombophilia who were identified as carrying a R338L missense mutation of FIX. 35 The plasma FIX activity of the proband was 800% of normal, and an rFIX-R338L exhibited a 5-to 10-fold higher specific activity in vitro. Hopfner et al 36 used insights from the structural and functional homology between FIXa and activated factor X (FXa) to bioengineer a recombinant FIX-FX hybrid that exhibited a catalytic efficiency 130-fold that of the wild-type rFIX.…”

Section: Increased Potencymentioning

confidence: 91%

Hemophilia: New Protein Therapeutics

Pipe

2010

Hematology

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Therapeutic advances for patients with hemophilia have resulted in reduced mortality, improved joint outcomes, safety from blood-transmitted pathogens, improved quality of life, and a normalized life span in the developed world. The production of recombinant coagulation factors has increased the worldwide capacity for replacement therapy and facilitated aggressive prophylactic therapy. However, this has come at significant cost, and barriers remain to broad application of prophylaxis. Recombinant DNA technology remains a promising platform to develop novel hemophilia therapeutics with improved functional properties to try to overcome some of these remaining barriers. Bioengineering strategies have produced novel therapeutics with increased production efficiency, increased potency and resistance to inactivation, prolonged plasma half-lives, and reduced immunogenicity. Alternative nonbiologic therapies may lead to new treatment paradigms. The current pipeline of new technologies and products is promising and growing with several agents already advancing from preclinical to clinical trials.

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X-Linked Thrombophilia with a Mutant Factor IX (Factor IX Padua)

Cited by 303 publications

References 21 publications

Computationally designed liver-specific transcriptional modules and hyperactive factor IX improve hepatic gene therapy

Computationally designed liver-specific transcriptional modules and hyperactive factor IX improve hepatic gene therapy

AAV liver expression of FIX-Padua prevents and eradicates FIX inhibitor without increasing thrombogenicity in hemophilia B dogs and mice

Hemophilia: New Protein Therapeutics

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