Piggybac Mediated Gene Transfer To Correct Hemophilia A

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2900-2900
Author(s):  
Janice M Staber ◽  
Molly Pollpeter ◽  
Angela Arensdorf ◽  
Patrick L Sinn ◽  
Thomas D Rutkowski ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Factor Viii ◽  
Hemophilia A ◽  
Viral Vector ◽  
Conflicts Of Interest ◽  
Vector System ◽  
Expression Vectors ◽  
Dna Repeats ◽  
Tail Clip

Abstract Hemophilia A, caused by a deficiency in factor VIII (FVIII), is the most severe inherited bleeding disorder, affecting about 1 out of 5,000 males; those affected suffer disabling joint and muscle hemorrhages. Hemophilia A is an attractive gene therapy candidate, because even small increases in FVIII levels (5-10%) will alter the phenotype. Non-viral vector systems are used increasingly in gene targeting technologies and as tools for gene transfer applications. Nonviral DNA transposons are genetic elements consisting of inverted terminal DNA repeats which in their naturally occurring configuration flank a transposase coding sequence. The transposase follows a “cut and paste” mechanism to excise the transposon from its original genomic location and insert it into a new locus. The insect derived piggyBac (PB) can be engineered to carry a therapeutic transgene between the inverted terminal repeats. Advantages of this novel nonviral vector system include a large transgene cassette capacity, ease of production and purification, and potential for site-specific integration. We hypothesize that a PB transposon vector carrying a codon-optimized human FVIII cDNA along with a hyperactive transposase (iPB7) will confer persistent gene expression and correction of the hemophilia A bleeding phenotype. We engineered PB transposon to carry a codon-optimized human FVIII B-domain deleted cDNA (coFVIII-BDD). We evaluated the in vivo gene transfer efficiency in hemophilia A mice by hydrodynamic tail-vein injection using PB coFVIII-BDD driven by the murine albumin enhancer/human alpha anti-trypsin promoter. Factor VIII null mice received 25 micrograms each of the PB coFVIII-BDD transposon and iPB7 to determine long term expression and phenotypic correction. FVIII activity and antigen levels were measured prior to injection and then every 4 weeks for 24 weeks. Results revealed therapeutic levels (50-225%) of factor VIII activity and antigen post gene transfer with stable expression for 24 weeks in most mice. A goal of gene transfer based therapies is to develop the most efficacious expression vectors with the least toxicity. To assess endoplasmic reticulum stress in the livers of treated and untreated mice, we evaluated BiP, CHOP, and EDEM levels via q-PCR. All experimental mice, null mice, and transposon treated mice without the coFVIII-BDD cassette revealed no evidence of cell stress. These data indicate codon-optimized FVIII and the piggyBac transposon vector system may provide a safe long term gene transfer strategy. To evaluate phenotypic correction, a tail clip assay was performed at the end of the study. More than 75% of mice receiving PB coFVIII-BDD transposon and iPB7 demonstrated functional correction via tail clip. These data show that the PB vector can be used to deliver transgene expression to the liver and achieve long term expression and phenotypic correction. Disclosures: No relevant conflicts of interest to declare.

A Gene Transfer Approach towards Hemophilia A Correction Using the Piggybac Transposon Vector.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1477-1477 ◽  
Author(s):  
Janice M. Staber ◽  
Erin R Burnight ◽  
Pavel Korsakov ◽  
Joseph Kaminski ◽  
Nancy L Craig ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Factor Viii ◽  
Cell Line ◽  
Hemophilia A ◽  
Vector System ◽  
Dna Repeats ◽  
Human Factor Viii ◽  
The Impact

Abstract Abstract 1477 Human Factor VIII (hFVIII) deficiency offers advantages as a disease target for gene therapy as small increases in factor VIII levels will alter the bleeding phenotype. In addition, both mouse and dog models of the disease are available for preclinical studies. Nonviral DNA transposons are genetic elements consisting of inverted terminal DNA repeats which in their naturally occurring configuration flank a transposase coding sequence. The transposase follows a “cut and paste” mechanism to excise the transposon from its original genomic location and insert it into a new locus. The insect derived piggyBac (PB) can be engineered to carry a therapeutic transgene between the inverted terminal repeats. Wu et al and others reported that piggyBac transposase is highly efficient at catalyzing transposition in mammalian cells in vitro (PNAS 103: 15008–15013, 2006). Advantages of this novel nonviral vector system include a large transgene cassette capacity and ease of production and purification. We hypothesize that a PB transposon vector carrying a reporter gene cassette or the human FVIII cDNA along with a codon-optimized (co-) or hyperactive (hyp-) transposase will confer persistent gene expression and correction of the hemophilia A bleeding phenotype with the FVIII cDNA. PB transposons were engineered to carry a puromycin resistance gene (PB puro), a human alpha1 antitrypsin reporter (PB hAAT), or hFVIII gene (B domain deleted or a partial B domain-226 amino acids/N6). We evaluated co- and hyp-transposase-mediated transposition in the Huh-7 human hepatoma cell line to verify function in hepatocytes. Using the PB puro vector, we demonstrated that the hyp-transposase generated a 2 fold higher transposition efficiency than the co-transposase in hepatocytes. We investigated the impact of varying the ratio of transposon to transposase; we screened ratios of 5:1, 2:1, 1:1, 1:2, and 1:5 in the Huh-7 cell line. Overall, the 1:2 and 1:1 ratios gave the greatest transposition efficiency in vitro. We evaluated the in vivo gene transfer efficiency in mice by hydrodynamic tail-vein injection using PB hAAT driven by the murine albumin enhancer/human alpha anti-trypsin promoter. Either a low (5 micrograms transposon) or high (25 micrograms transposon) dose was given with varying amounts of hyp-transposase to generate an in vivo dose response curve. Serum hAAT levels were measured prior to injection and then monthly for 3 months. Results revealed the 1:1 ratio at the high transposon dose generated higher level of expression compared to all other doses with expression stable in all groups for 3 months. PB vectors encoding hFVIII have been prepared, and our studies with these vectors are ongoing. These data show that the PB vector can be used to deliver transgene expression to the liver and achieve long term expression of a secreted protein. Disclosures: Staber: Bayer Healthcare: Research Funding.

Clotting Factor VIII Overexpression Shows Signs of ER Stress but Does Not Cause Toxicity upon Gene Transfer

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3238-3238
Author(s):  
Irene Zolotukhin ◽  
Brett Palaschak ◽  
David M Markusic ◽  
Roland Herzog
Keyword(s):  
Stress Response ◽  
Gene Transfer ◽  
Factor Viii ◽  
Er Stress ◽  
Hemophilia A ◽  
Conflicts Of Interest ◽  
Clotting Factor ◽  
Potent Inducer ◽  
Cost Effective Treatment ◽  
Human Factor Ix

Abstract Hemophilia A is the X-linked bleeding disorder resulting from the loss of functional clotting factor VIII (FVIII). Hemophilia A patients with severe disease (<1% residual FVIII activity) experience spontaneous bleeds into the joints and closed spaces with severe morbidity. Restoration of hemostasis is managed by repeated infusions (2-3 times per week) of plasma derived or recombinant FVIII protein. While a standard treatment is available for patients, life-long infusions of FVIII protein is very expensive, has a negative impact on the patient's quality of life, and FVIII protein products are not available worldwide. Hence, there is a need to develop a more robust and cost effective treatment for hemophilia A patients. Liver-directed gene therapy using adeno-associated virus (AAV) represents a promising approach to treat hemophilia A. However, previous studies have shown that overexpression of human FVIII protein in the context of hydrodynamic delivery of plasmid vectors induces ER stress mediated through the unfolded protein response (UPR). Because human FVIII protein is inefficiently secreted into circulation, high AAV vector doses will be required to obtain therapeutic expression levels. Therefore, we sought to determine if AAV-FVIII gene delivery also triggers cellular UPR in hepatocytes in vivo. To this end, we selected to use a codon-optimized FVIII cDNA, which has been shown by our lab to significantly increase FVIII protein expression, and a high vector dose of AAV8-ApoE-hAAT-cohF8, containing a hepatocyte-specific enhancer/promoter combination. We evaluated this vector at doses of 1 x 1011 vg (4x1012 vg/kg) and 1 x 1012 vg (4x1013 vg/kg) in hemophilia A mice on a 129/BL6 background. Intravenous administration of the highest vector dose completely restored hemostasis, which was sustained and achieved super-physiological levels in some animals. Importantly, none of these mice developed inhibitors against FVIII. Next, we administered the vector at the same 2 doses to C67BL/6 mice, which show higher hepatic transduction efficiency than other strains. Experimental controls consisted of mice, with no vector or 1x1012 vg of AAV8-ApoE-hAAT-F9, expressing human factor IX (FIX) protein. Injection of tunicamycin, a potent inducer of the ER stress response, served as a positive control for all assays. Vector-treated mice were studied 2 and 4 weeks after gene transfer (n=3-4 per group). First, we evaluated the status of key molecular chaperones, known to be the mediators of the UPR: Bip, p-PERK, and p-eIF2a. Western blotting performed on the liver lysates indicated modest up-regulation of all three markers compared to normal control, but that effect was neither dose nor gene dependent. In addition, we tested the splicing of Xbp1 mRNA by PCR assay and observed low level of the 26 bp spliced fragment, indicative of the UPR, at the high vector dose. Immunohistochemistry on liver sections from each of our experimental groups including H&E staining, Tunnel staining for apoptotic cells, and reactive oxygen species staining. None of the stains yielded evidence for liver damage even in the 1x1012 AAV8-cohF8 treated mice compared to untreated controls. There was also no elevation of liver enzyme levels in plasma samples. Analysis of plasma from vector injected mice showed systemic levels of human FVIII and FIX proteins at ~30 ng/ml and ~6300 ng/ml, respectively (these ELISA-based measurements likely underestimate FVIII levels due to interference by von Willebrand factor). Overall our results suggest that over-expression of coagulation factors in hepatocytes from AAV vectors causes a mild cell stress response that is not strong enough to cause liver toxicity, is not specific for FVIII, and does impact expression or immunogenicity. Disclosures No relevant conflicts of interest to declare.

Gene Transfer Targeting Hepatocytes Using the PiggyBac Transposon System: An Approach towards Hemophilia A Correction and Long Term Expression of Factor VIII.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3575-3575
Author(s):  
Janice M. Staber ◽  
Erin Burnight ◽  
Marie- Ellen Sarvida ◽  
Anton McCaffrey ◽  
Joseph Kaminski ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Factor Viii ◽  
Hemophilia A ◽  
Vector System ◽  
Luciferase Expression ◽  
Post Injection ◽  
Piggybac Transposon ◽  
Tail Vein ◽  
Tail Vein Injection

Abstract Abstract 3575 Poster Board III-512 Human Factor VIII (hFVIII) deficiency offers advantages as a disease target for gene therapy as small increases in factor VIII levels will alter the bleeding phenotype. In addition, both mouse and dog models of the disease are available for preclinical studies. Nonviral DNA transposons are genetic elements consisting of inverted terminal DNA repeats which in their naturally occurring configuration flank a transposase coding sequence. The transposase follows a “cut and paste” mechanism to excise the transposon from its original genomic location and insert it into a new locus. The insect derived piggyBAC can be engineered to carry a therapeutic transgene between the inverted terminal repeats. Wu et al and others reported that piggyBAC transposase is highly efficient at catalyzing transposition in mammalian cells in vitro (PNAS 103: 15008-15013, 2006). To date, there are no published reports of in vivo gene transfer to mammalian livers using the piggyBAC transposon system. Advantages of this novel nonviral vector system include a large transgene cassette capacity, ease of production and purification, and the ability to excise itself precisely without leaving a footprint. We hypothesize that a piggyBAC transposon vector carrying a reporter gene cassette or the human FVIII cDNA along with a codon-optimized (co-) transposase will confer persistent gene expression and correction of the hemophilia A bleeding phenotype with the FVIII cDNA. PiggyBAC transposons were engineered to carry a hygromycin resistance gene (Hygro), a luciferase expression cassette (PB luciferase), or a human alpha1 antitrypsin reporter (hAAT). We evaluated co- transposase-mediated transposition in the Huh-7 human hepatoma cell line to verify function in hepatocytes. Using the PB hygro vector, we demonstrated that the co- transposase generated higher transposition efficiency than an inactive mutant in hepatocytes. We then showed in vivo persistence following hydrodynamic tail-vein injection using firefly luciferase expression driven by the murine albumin enhancer/human alpha anti-trypsin promoter. Luciferase expression measured via in vivo bioluminescence imaging persisted up to eight months in C57Bl/6 liver (duration of experiment). Following partial hepatectomies at 5 months post injection, expression was observed only in animals receiving PB luciferase transposon and an active transposase while expression in those treated with the inactive mutant dropped to background levels supporting that expression was from integrated transgene. We furthered these experiments by introducing PB hAAT via hydrodynamic tail-vein injection as before at either a low (12.5 micrograms each transposon and transposase) or high (50 micrograms each) dose. Serum hAAT levels were measured at 421ng/ml and 365ng/ml via ELISA at 3 months post-injection, respectively. PB vectors encoding hFVIII have been prepared, and our studies with these vectors are ongoing. These data represent one of the first studies to show persistent transgene expression in vivo from piggyBAC transposon gene transfer. Disclosures: No relevant conflicts of interest to declare.

Bioengineered Coagulation Factor VIII Enables Long-Term Correction Of Murine Hemophilia A Following Liver-Directed Adeno-Associated Viral Vector Delivery

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4210-4210
Author(s):  
Harrison C. Brown ◽  
Jordan E Shields ◽  
Shangzhen Zhou ◽  
J. Fraser Wright ◽  
H. Trent Spencer ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Factor Viii ◽  
Hemophilia A ◽  
Viral Vector ◽  
Coagulation Factor ◽  
High Expression ◽  
Female Mice ◽  
Equity Ownership ◽  
Packaging Capacity

Abstract Preclinical and, more recently, clinical data support the feasibility and safety of recombinant adeno-associated viral vectors (rAAV) in gene therapy applications. Despite several clinical trials of rAAV-based gene transfer for hemophilia B, a unique set of obstacles impede the development of a similar approach for hemophilia A. These include 1) inefficient biosynthesis of human coagulation factor VIII (fVIII), 2) limited packaging capacity of rAAV (4.9kb) for the large B domain-deleted (BDD) fVIII transgene (4.5 kb), 3) humoral immune responses to the transgene product and 4) dose limitations imposed by capsid mediated cytotoxic immunity. Our laboratory has developed and validated bioengineered fVIII molecules that are biosynthesized more efficiently than similar BDD human fVIII molecules due to superior post-translational transit through the ER/golgi/secretory pathway. In the current study, we incorporated one of these constructs, previously designated HP47 (Doering et al. 2004 J Biol Chem. 279:8) and now termed ET-3, into an AAV-based gene transfer approach similar to that previous tested for patients with hemophilia B. Briefly, a rAAV2/8 vector encoding the ET-3 transgene under the control of a liver-specific promoter comprised of an Apo E hepatic control region (HCR) and the α-1 antitrypsin enhancer/promoter (HAAT), flanked by AAV inverted terminal repeats (ITRs), was constructed and designated rAAV-HCR-ET3. In prior studies, we demonstrated that recombinant ET-3 displays i) 100-fold more efficient biosynthesis, ii) 2 – 3-fold higher specific activity, and iii) 3-fold slower decay compared to BDD human fVIII. However, the vector genome size of rAAV-HCR-ET3, 5.9kb, exceeds the packaging capacity of rAAV by 20%. To address this concern, molecular analysis of rAAV-HCR-ET3 was performed and showed packaging primarily of 5' or 3' truncated vector genomes (vg). Further quantitative (q) PCR of vg using a series of primers pairs spanning the vector sequence showed 5-fold fewer copies of the termini than the center, which is indicative of terminally truncated vg. Thus, special consideration must be taken when using qPCR-based methods alone to titer vector preparations. Our data support a model whereby cells are infected by multiple truncated but overlapping vg, which then reassemble at some frequency to form complete, functional fVIII transgenes and/or RNA transcripts. In the present study, adult hemophilia A mice were administered a single peripheral vein injection of rAAV-HCR-ET3 at doses ranging from 3.9e10 vector particles (vp) /kg to 2.0e13 vp/kg as determined by viral protein concentration. These stated doses are 5 – 25-fold higher than would have been assigned using qPCR and expressed as vg/kg as opposed to vp/kg. At the conclusion of the study (11 - 50 weeks post-infusion), correction of fVIII deficiency to curative levels of fVIII (>0.4 U/mL in circulating plasma) was achieved at vector doses as low as 6.25e11 vp/kg, and partial correction (>0.01 U/mL) was seen at doses as low as 1.6e11 vp/kg. Tail transection bleeding assay demonstrated correction of the bleeding phenotype out to as long as 50 weeks after rAAV infusion. While gene transfer and phenotypic correction was achieved in both male and female mice, male mice receiving 2.5e12 vp/kg showed 9-fold greater circulating fVIII levels than female mice receiving the same dose, which is consistent with previous reports. Taken together, these data suggest significant benefit of the bioengineered high expression fVIII transgene (ET-3) in the context of liver-directed rAAV delivery at low viral vector doses, which if based on qPCR, could be stated as low as 1e10 vg/kg and even that includes mostly incomplete vg. Recent data suggest that reducing the size of the rAAV-fVIII vg through the incorporation of shorter DNA control elements and smaller fVIII transgenes can increase the vector potency (McIntosh et al. 2013 Blood 121:17). Despite its large vg size, rAAV-HCR-ET3 achieved curative levels of fVIII activity at vector doses comparable to those recently reported for smaller vectors. Therefore, it appears likely that further enhancement of the rAAV-bioengineered high expression fVIII gene transfer approach will be possible through incorporation of smaller genetic elements that increase the frequency of functional ET-3 gene transfer in the context of hemophilia A gene therapy. Disclosures: Spencer: Expression Therapeutics, LLC: Equity Ownership. Doering:Expression Therapeutics, LLC: Equity Ownership.

scholarly journals Long-Term Expression of Von Willebrand Factor Via Piggybac-Mediated Gene Transfer

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3511-3511 ◽  
Author(s):  
Blake A Johnson ◽  
Anil K. Chauhan ◽  
Janice M Staber
Keyword(s):  
Gene Transfer ◽  
Von Willebrand Factor ◽  
Von Willebrand Disease ◽  
Vector System ◽  
Transfer Strategies ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Tail Clip

Abstract von Willebrand disease (vWD), caused by a deficiency in von Willebrand factor (vWF), is the most common inherited bleeding disorder, affecting up to 1% of the population; those affected have a varied bleeding phenotype. von Willebrand factor plays two important roles in hemostasis including platelet adhesion and stability of factor VIII (FVIII). As such, patients with von Willebrand disease type 3 have no measurable vWF protein and demonstrate low levels of FVIII. To date, persistent long-term expression of full-length vWF using gene transfer strategies has not been demonstrated, largely due to the immense size of vWF cDNA (8.4 kb). Non-viral vector systems, such as piggyBac, are used increasingly in gene targeting technologies and as tools for gene transfer applications. Nonviral DNA transposons are genetic elements consisting of inverted terminal DNA repeats which in their naturally occurring configuration flank a transposase coding sequence. The transposase follows a "cut and paste" mechanism to excise the transposon from its original genomic location and insert it into a new locus. The insect derived piggyBac (PB) can be engineered to carry a therapeutic transgene between the inverted terminal repeats. Advantages of this nonviral vector system include a large transgene cassette capacity, ease of production and purification, and potential for site-specific integration. We hypothesize PB-mediated vWF gene transfer will confer long-term expression and improve the bleeding phenotype in an animal model of vWD. We engineered PB transposon to carry a human von Willebrand factor cDNA (PB vWF). When transfected with PB-vWF and a hyperactive transposase, iPB7, the hepatocarcinoma cell line, HepG2, demonstrated secretion of vWF into supernatants compared to mock transfected controls (p < 0.01). Next, we evaluated the in vivo gene transfer efficiency in von Willebrand deficient mice by hydrodynamic tail-vein injection using PB-vWF driven by the murine albumin enhancer/human alpha anti-trypsin promoter. vWF null mice received 25 or 50 micrograms each of the PB-vWF transposon and iPB7 to determine long-term expression and phenotypic correction. vWF levels were measured prior to injection and then every 4 weeks for up to 24 weeks. Results revealed therapeutic levels (on average 50% normal mice) of vWF post gene transfer with stable expression for 24 weeks in most mice. These data indicate the PB transposon vector system may provide a long-term gene transfer strategy for vWD. To evaluate phenotypic correction, a tail clip assay was performed at the end of the study. PB-vWF gene transfer resulted in at least partial bleeding phenotype correction via tail clip. Additionally, treated mice demonstrated a rescue of the FVIII activity. These data show that the PB vector can be used to deliver this large transgene expression cassette to the liver and achieve long-term expression and phenotypic correction in vivo. Disclosures Staber: Emergent BioSolutions: Honoraria; Baxalta: Honoraria.

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