The efficacy and the risk of immunogenicity of FIX Padua (R338L) in hemophilia B dogs treated by AAV muscle gene therapy

Finn, Jonathan D.; Nichols, Timothy C.; Svoronos, Nikolaos; Merricks, Elizabeth P.; Bellenger, Dwight A.; Zhou, Shangshen; Simioni, Paolo; High, Katherine A.; Arruda, Valder R.

doi:10.1182/blood-2012-06-440123

Cited by 100 publications

(113 citation statements)

References 20 publications

Supporting

Mentioning

102

Contrasting

Order By: Relevance

“…In combination with a codon-usage optimized canine FIX, the FIX Padua mutation allowed an increase of the FIX activity to 125% of the physiological clotting levels resulting in 15-fold gain of gene therapy potency. Longtime correction of the bleeding phenotype was demonstrated in a canine model by expression of the gain-of-function FIX-R338L variant [45]. The transvenular AAV vector mediated delivery of FIX Padua resulted in circulating cFIX levels with a magnitude of expression which was similar to those in earlier observed studies with hemophilia B dogs using wt-cFIX [46].…”

Section: Engineering Fix Variants With Enhanced Specific Activitysupporting

confidence: 73%

“…The transvenular AAV vector mediated delivery of FIX Padua resulted in circulating cFIX levels with a magnitude of expression which was similar to those in earlier observed studies with hemophilia B dogs using wt-cFIX [46]. However the expression of FIX-R338L in the muscles of treated dogs resulted in 8-9-fold higher specific activity and normalization of the whole blood clotting time which was stable throughout the experiment followed up to 5 years [45]. Currently, these encouraging preclinical reports led to the recruitment of clinical gene therapy trial with the FIX Padua based on AAV serotype 8 gene delivery system into the liver (Registration number: NTC01687608).…”

Section: Engineering Fix Variants With Enhanced Specific Activitysupporting

confidence: 64%

See 1 more Smart Citation

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

2012

J Genet Syndr Gene Ther

View full text Add to dashboard Cite

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...

show abstract

Section: Engineering Fix Variants With Enhanced Specific Activitysupporting

confidence: 73%

Section: Engineering Fix Variants With Enhanced Specific Activitysupporting

confidence: 64%

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

2012

J Genet Syndr Gene Ther

View full text Add to dashboard Cite

show abstract

“…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). These FIX activity levels represent a robust improvement compared with the state-of-the-art AAV-FIX vectors, including those used in the most recent scAAV8-based clinical trial.…”

Section: Resultsmentioning

confidence: 99%

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

Nair¹,

Rincón

Evens³

et al. 2014

Blood

View full text Add to dashboard Cite

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...

show abstract

“…24 Canine FIX antigen, activity, and antibody assays Whole blood clotting time (WBCT), cFIX antigen and activity, and thromboelastography (TEG) were assayed as previously described. 25,26 Neutralizing antibodies to cFIX were determined by the Bethesda assay.…”

Section: Systemic and Local Toxicitymentioning

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

Self Cite

139

178

View full text Add to dashboard Cite

• 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

show abstract

The efficacy and the risk of immunogenicity of FIX Padua (R338L) in hemophilia B dogs treated by AAV muscle gene therapy

Cited by 100 publications

References 20 publications

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

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

Contact Info

Product

Resources

About