Hyperfunctional coagulation factor IX improves the efficacy of gene therapy in hemophilic mice

Cantore, Alessio; Nair, Nisha; Valle, Patrizia Della; Matteo, Mario Di; Mátrai, Janka; Sanvito, Francesca; Brombin, Chiara; Serio, Clelia Di; D’Angelo, Armando; Chuah, Marinee; Naldini, Luigi; VandenDriessche, Thierry

doi:10.1182/blood-2012-05-432591

Cited by 87 publications

(133 citation statements)

References 18 publications

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“…In contrast, an activity:antigen ratio equal to 1 was observed for the co-hFIX control at all time points and vector doses ( Figure 2J-L). This is consistent with our previous results using integrationcompetent and integration-defective lentiviral vectors 7 and with the increased FIX activity after muscle-directed AAV transduction in mouse and dog models. 13 Most importantly, even at the lowest vector dose tested (5 3 10 10 vg/kg), it was possible to obtain supraphysiologic FIX levels that corrected the bleeding diathesis (supplemental Figure 2A).…”

Section: Resultssupporting

confidence: 82%

“…5 Generation and initial characterization of the codon-optimized FIX (coFIX) with the hyperactivating Padua mutation (ie, coFIX-R338L) were described previously. 7 The most robust HS-CRM (designated as HS-CRM8) was cloned upstream of a minimal transthyretin promoter (TTR) that drives the coFIX-R338L and was incorporated into a self-complementary AAV (scAAV) vector (kindly provided by Dr Srivastava, University of Florida College of Medicine). 8 Alternatively, a green fluorescent protein (GFP) reporter gene was used to verify the tissuespecific expression patterns.…”

Section: Methodsmentioning

confidence: 99%

“…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%

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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: Resultssupporting

confidence: 82%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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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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“…Recent studies implementing this hyperfunctional FIX. Padua, also demonstrated its potential to correct the bleeding phenotype by allowing a reduction of the administered lentiviral vector dose for liver targeted expression in mice [44]. In this study the introduction of R338L in canine FIX led to 5-fold higher activity compared to canine wt-FIX.…”

Section: Engineering Fix Variants With Enhanced Specific Activitymentioning

confidence: 75%

Engineered Factor VII, Factor IX, and Factor X Variants for Hemophilia Gene Therapy

2012

J Genet Syndr Gene Ther

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FVII/FVIIaPatients with hemophilia A and B have deficient protein concentrations of FVIII and FIX, respectively and therefore are treated by protein substitution with plasma-derived or recombinant factor FVIII or FIX concentrates. In about 20-30% of the patients with hemophilia A and in 3-5% of those with hemophilia B this can lead to the development of neutralizing antibodies against the infused proteins which makes therapy inefficient [1,2]. Recombinant activated factor VII (rFVIIa) protein is currently an alternative therapy available for inhibitor patients to treat excessive bleeding. However, supraphysiological concentrations and repeated administration are required to induce hemostasis [3].Consequently, FVIIa analogs were created for future protein replacement therapy or gene delivery approaches based on crystalline structure analysis of free and TF-bound FVIIa or on sequence homology of other more potent proteases [4,5].Like the other vitamin K dependent coagulation factors, FVII is physiologically synthesized in the liver. FVII is secreted into the circulation as a 406-residue single-chain polypeptide which contains an N-terminal γ-carboxyglutamic acid (Gla) domain in which all ten Glu residues are post-translationally carboxylated followed by two regions homologous to epidermal growth factor domains and a serine protease domain [3,6,7]. Upon vascular injury, the cofactor tissue factor (TF) is exposed to FVII and triggers the extrinsic pathway of the coagulation cascade by activation of FIX, factor X and FVII auto-activation on the TF-bearing cells resulting in large-scale thrombin generation and a fibrin clot. FVII is activated to FVIIa by internal proteolysis due to a cleavage of a single Arg152-Ile153 peptide bond. The physiological plasma concentration of FVII is around 10 nM, however, only about 1% of FVII is circulating in its free and active form. FVIIa has a plasma halflife of approximately 2.5 hours [8]. FVII activation results in connected FVII light and heavy chains which form a one-to-one complex with TF in the presence of Ca 2+ ions. The complex formation is an essential process in which TF allosterically leads to a fully active FVIIa [9]. The cleavage alone leaves FVIIa in a zymogen-like conformation of quite low specific activity [3,10]. Several residues in the first EGF-like domain of FVII and in the protease domain, especially methionine at position 306, seem to be pivotal for the cofactor-mediated allosteric stimulation [11]. TF stabilizes the active conformation of FVIIa for accelerated activation of its substrates FIX and FX [12,13] by inducing a conformational change and establishing a stable salt bridge between the residues Ile153 and Asp343 [14] and stabilization of the interaction between the residues Leu305 and Phe374 which in turn stabilizes S 1 and S 3 substrate pocket and the activation pocket [15]. These findings contributed to the development of FVIIa variants with enhanced TFindependent (intrinsic) specific activity by mimicking the effect of TF binding [16]. Additional fin...

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“…Inclusion of this modified FIX has been shown to improve vector and transgene potency in preclinical AAV gene therapy studies. 19,20 Collectively, evidence is building to support FVIII and FIX transgene engineering approaches as critical components to the success of stem cell-based gene therapies for hemophilia A and B. However, the safety and efficacy of these molecules remain to be tested and proven in clinical trials.…”

Section: Origins and Limitations Of Fviii And Fix Biosynthesismentioning

confidence: 99%

Replacing bad (F)actors: hemophilia

Doering

Spencer

2014

Hematology

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Hemophilia A and B are bleeding disorders that result from functional deficiencies in specific circulating blood clotting factors termed factor VIII (FVIII) and factor IX (FIX), respectively, and collectively display an incidence of 1 in 4000 male births. Stem cell transplantation therapies hold the promise of providing a cure for hemophilia, but currently available transplantable stem cell products do not confer endogenous FIX or FVIII biosynthesis. Learning Objective• To gain an understanding of the key issues surrounding the implementation of stem cell therapies for hemophilia

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Hyperfunctional coagulation factor IX improves the efficacy of gene therapy in hemophilic mice

Cited by 87 publications

References 18 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

Engineered Factor VII, Factor IX, and Factor X Variants for Hemophilia Gene Therapy

Replacing bad (F)actors: hemophilia

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