Transgene Loss and Changes in the Promoter Methylation Status as Determinants for Expression Duration in Nonviral Gene Transfer for Factor IX

Schüttrumpf, Jörg; Milanov, Peter; Abriss, Daniela; Roth, Stefanie; Tonn, Torsten; Seifried, Erhard

doi:10.1089/hum.2009.212

Cited by 16 publications

(18 citation statements)

References 16 publications

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“…32 However, Schüttrumpf et al . 33 recently showed that the predominant reason for transient expression after hydrodynamic infusion in mouse liver was loss of the vector, regardless of whether it was a plasmid or MC construct. Nevertheless, they observed a higher expression from the remaining MCs, which is in line with our findings; we observe equal amounts of remaining vector in the miMC and plasmid groups but higher expression and effect from the miMC.…”

Section: Discussionmentioning

confidence: 99%

Micro-minicircle Gene Therapy: Implications of Size on Fermentation, Complexation, Shearing Resistance, and Expression

Stenler

Wiklander

Badal-Tejedor³

et al. 2014

Molecular Therapy - Nucleic Acids

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The minicircle (MC), composed of eukaryotic sequences only, is an interesting approach to increase the safety and efficiency of plasmid-based vectors for gene therapy. In this paper, we investigate micro-MC (miMC) vectors encoding small regulatory RNA. We use a construct encoding a splice-correcting U7 small nuclear RNA, which results in a vector of 650 base pairs (bp), as compared to a conventional 3600 bp plasmid carrying the same expression cassette. Furthermore, we construct miMCs of varying sizes carrying different number of these cassettes. This allows us to evaluate how size influences production, super-coiling, stability and efficiency of the vector. We characterize coiling morphology by atomic force microscopy and measure the resistance to shearing forces caused by an injector device, the Biojector. We compare the behavior of miMCs and plasmids in vitro using lipofection and electroporation, as well as in vivo in mice. We here show that when the size of the miMC is reduced, the formation of dimers and trimers increases. There seems to be a lower size limit for efficient expression. We demonstrate that miMCs are more robust than plasmids when exposed to shearing forces, and that they show extended expression in vivo.

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Section: Discussionmentioning

confidence: 99%

Micro-minicircle Gene Therapy: Implications of Size on Fermentation, Complexation, Shearing Resistance, and Expression

Stenler

Wiklander

Badal-Tejedor³

et al. 2014

Molecular Therapy - Nucleic Acids

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“…FIX-deficient mice on a C57Bl/6 background were kindly provided by Katherine High [25]. Minicircle DNA was injected hydrodynamically via the tail vein as previously described [26] and 2% (w/v) anhydrous betaine was administered solubilized in drinking water ad libitum for defined time periods: Drinking water was replenished at least every 48 h. FVIII knockout mice received hydrodynamic injections of 20 µg of Minicircle-DNA expressing hFVIII-BDD or FVIII-BDDQ305P. Using a cross-over design, 24 hours following gene transfer, mice were randomly assigned to the control or the betaine-treated group for 3 days, then plasma samples were retroorbitally collected and treatment was switched between the groups for another 3 day-interval.…”

Section: Methodsmentioning

confidence: 99%

Chemical Chaperones Improve Protein Secretion and Rescue Mutant Factor VIII in Mice with Hemophilia A

et al. 2012

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Inefficient intracellular protein trafficking is a critical issue in the pathogenesis of a variety of diseases and in recombinant protein production. Here we investigated the trafficking of factor VIII (FVIII), which is affected in the coagulation disorder hemophilia A. We hypothesized that chemical chaperones may be useful to enhance folding and processing of FVIII in recombinant protein production, and as a therapeutic approach in patients with impaired FVIII secretion. A tagged B-domain-deleted version of human FVIII was expressed in cultured Chinese Hamster Ovary cells to mimic the industrial production of this important protein. Of several chemical chaperones tested, the addition of betaine resulted in increased secretion of FVIII, by increasing solubility of intracellular FVIII aggregates and improving transport from endoplasmic reticulum to Golgi. Similar results were obtained in experiments monitoring recombinant full-length FVIII. Oral betaine administration also increased FVIII and factor IX (FIX) plasma levels in FVIII or FIX knockout mice following gene transfer. Moreover, in vitro and in vivo applications of betaine were also able to rescue a trafficking-defective FVIII mutant (FVIIIQ305P). We conclude that chemical chaperones such as betaine might represent a useful treatment concept for hemophilia and other diseases caused by deficient intracellular protein trafficking.

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“…14,23 To examine whether the introduced amino acid substitutions lead to novel immunogenic epitopes in FIX, BALB/c mice were either not previously treated or were made (BeneFIX); and lane 6, hFIXa control. Zymogen or FIXa was detected using an anti-hFIX mAb directed toward the heavy chain of hFIX.…”

Section: Fix Variants Do Not Cause Immune Responses In Fix-wt-toleranmentioning

confidence: 99%

“…This immunoassay, which was developed to detect human TAT, is highly cross-reactive to murine TAT complexes, as described previously. 13 Abs to hFIX were measured by a specific ELISA to murine Ig subclasses (IgG1, IgG2a, IgG2b, IgG3, IgM, and IgA), as described previously 14 with minor modifications, by coating plates with 1 g/mL of purified recombinant hFIX (BeneFIX; Pfizer) or the hFIX variants T or ITV. We tested for functionally inhibitory Abs against hFIX-WT and hFIX variants with a modified Bethesda assay in which we mixed plasma samples from BALB/c mice with equal volumes of hFIX-WT or variants at different dilutions.…”

Section: Fix and Coagulation Assaysmentioning

confidence: 99%

Engineered factor IX variants bypass FVIII and correct hemophilia A phenotype in mice

Milanov

Ivanciu

Abriss

et al. 2012

Blood

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The complex of the serine protease factor IX (FIX) and its cofactor, factor VIII (FVIII), is crucial for propagation of the intrinsic coagulation cascade. Absence of either factor leads to hemophilia, a disabling disorder marked by excessive hemorrhage after minor trauma. FVIII is the more commonly affected protein, either by X-chromosomal gene mutations or in autoimmune-mediated acquired hemophilia. Whereas substitution of FVIII is the mainstay of hemophilia A therapy, treatment of patients with inhibitory Abs remains challenging. In the present study, we report the development of FIX variants that can propagate the intrinsic coagulation cascade in the absence of FVIII. FIX variants were expressed in FVIII-knockout (FVIII-KO) mice using a nonviral genetransfer system. Expression of the variants shortened clotting times, reduced blood loss after tail-clip assay, and reinstalled clot formation, as tested by in vivo imaging of laser-induced vessel injury. In IntroductionThe intrinsic coagulation cascade is a tightly regulated proteaseand cofactor-dependent amplification system that ensures the formation of stable clots after injury. 1 Within this system, deficiencies of the coagulation cofactor, factor VIII (FVIII), or the corresponding coagulation protease, factor IX (FIX), lead to the X-chromosomal inherited bleeding disorders hemophilia A and hemophilia B, respectively. Hemophilia A occurs approximately in 1 of 5000 newborn boys, whereas hemophilia B is less common. 2 Untreated, hemophilia presents with spontaneous bleeding preferentially into large joints and skeletal muscle, and internal and intracranial bleedings can also occur. Substitution of deficient coagulation factors by intravenous infusion of plasma-derived or recombinant coagulation factor concentrates is the therapy of choice. In the last decades, treatment has evolved from so-called on-demand treatment for acute injury/hemorrhage to a secondary preventative approach with regular prophylactic infusions. Prophylactic treatment, securing plasma levels above 1% of normal, already prevents the major long-term consequences of the disease: joint damage and muscular atrophy. 3,4 A major obstacle for protein substitution therapy is the occurrence of neutralizing Abs directed against the FVIII protein. This can either be a result of an immune response after exogenous protein exposure 5 or may appear in adult patients as a spontaneous auto-immune event. 6 In these cases, FVIII infusion is often ineffective and so-called bypassing agents are used. These agents consist of constitutively activated proteases such as activated factor VII (FVIIa), and promote clot formation directly without restoring the intrinsic amplification loop. Although these therapeutics are efficient at stopping acute bleeding, limitations include the relatively short half-lives of activated proteases in the circulation and potential vascular risks in long-term treatment. 7,8 In the present study, we report on the generation of FIX variants with FVIII-independent clotting activity that we...

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Transgene Loss and Changes in the Promoter Methylation Status as Determinants for Expression Duration in Nonviral Gene Transfer for Factor IX

Cited by 16 publications

References 16 publications

Micro-minicircle Gene Therapy: Implications of Size on Fermentation, Complexation, Shearing Resistance, and Expression

Micro-minicircle Gene Therapy: Implications of Size on Fermentation, Complexation, Shearing Resistance, and Expression

Chemical Chaperones Improve Protein Secretion and Rescue Mutant Factor VIII in Mice with Hemophilia A

Engineered factor IX variants bypass FVIII and correct hemophilia A phenotype in mice

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