Ultrasound-mediated gene delivery of factor VIII plasmids for hemophilia A gene therapy in mice

IntroductionHemophilia A results from the plasma deficiency of factor VIII, a gene carried on the X chromosome. Bleeding results from a lack of coagulation factor VIII, a large and complex protein that circulates in complex with its carrier, von Willebrand factor (vWF).1 Severe hemophilia A (<1% of normal circulating levels) is associated with a high degree of mortality, due to spontaneous and trauma-induced, life-threatening and crippling bleeding episodes.2 Current treatment in the United States consists of infusion of plasma-derived or recombinant factor VIII in response to bleeding episodes.3 Such treatment fails to prevent cumulative joint damage, a major cause of hemophilia-associated morbidity.4 Availability of prophylactic treatment, which would reduce the number and severity of bleeding episodes and, consequently, would limit such joint damage, is limited by cost and the problems associated with repeated venous access. Other problems are associated with frequent replacement treatment, including the dangers of transmission of blood-borne infections derived from plasma used as a source of factor VIII or tissue culture or formulation components. These dangers are reduced, but not eliminated, by current manufacturing techniques. Furthermore, approximately 1 in 5 patients with severe hemophilia treated with recombinant or plasma-derived factor VIII develop inhibitory humoral immune responses. In some cases, new inhibitors have developed, apparently in response to unnatural modifications introduced during manufacture or purification.5 Gene therapy could circumvent most of these difficulties. In theory, a single injection of a vector encoding the factor VIII gene could provide constant plasma levels of factor in the long term. However, long-term expression after gene transfer of a systemically expressed protein in higher mammals has seldom been described. In some cases, a vector that appeared promising in a rodent model has not worked well in larger animals, for example, due to a massive immune response not seen in the rodent.6 An excellent review of early efforts at factor VIII gene therapy appeared in an earlier volume of this series.7 A summary of results from various in vivo experiments is shown in Table 1. This chapter will focus on results pertaining to studies using vectors based on murine retroviruses, including our own work.

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Codon optimization of human factor VIII cDNAs leads to high-level expression

Blood ◽

10.1182/blood-2010-05-282707 ◽

2011 ◽

Vol 117 (3) ◽

pp. 798-807 ◽

Cited By ~ 109

Author(s):

Natalie J. Ward ◽

Suzanne M. K. Buckley ◽

Simon N. Waddington ◽

Thierry VandenDriessche ◽

Marinee K. L. Chuah ◽

...

Keyword(s):

Gene Therapy ◽

Factor Viii ◽

Viral Vectors ◽

Hemophilia A ◽

Lentiviral Vector ◽

Wild Type ◽

Fviii Activity ◽

Human Factor Viii

Abstract Gene therapy for hemophilia A would be facilitated by development of smaller expression cassettes encoding factor VIII (FVIII), which demonstrate improved biosynthesis and/or enhanced biologic properties. B domain deleted (BDD) FVIII retains full procoagulant function and is expressed at higher levels than wild-type FVIII. However, a partial BDD FVIII, leaving an N-terminal 226 amino acid stretch (N6), increases in vitro secretion of FVIII tenfold compared with BDD-FVIII. In this study, we tested various BDD constructs in the context of either wild-type or codon-optimized cDNA sequences expressed under control of the strong, ubiquitous Spleen Focus Forming Virus promoter within a self-inactivating HIV-based lentiviral vector. Transduced 293T cells in vitro demonstrated detectable FVIII activity. Hemophilic mice treated with lentiviral vectors showed expression of FVIII activity and phenotypic correction sustained over 250 days. Importantly, codon-optimized constructs achieved an unprecedented 29- to 44-fold increase in expression, yielding more than 200% normal human FVIII levels. Addition of B domain sequences to BDD-FVIII did not significantly increase in vivo expression. These significant findings demonstrate that shorter FVIII constructs that can be more easily accommodated in viral vectors can result in increased therapeutic efficacy and may deliver effective gene therapy for hemophilia A.

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Direct Comparison of Bioengineered Factor VIII Expression Constructs: Towards an Optimal Transgene for Gene Therapy of Hemophilia A.

Blood ◽

10.1182/blood.v108.11.5477.5477 ◽

2006 ◽

Vol 108 (11) ◽

pp. 5477-5477

Author(s):

Kerry L. Titus ◽

Paul Lee ◽

H. Trent Spencer ◽

Christopher Doering

Keyword(s):

Gene Therapy ◽

Amino Acid ◽

Factor Viii ◽

Hemophilia A ◽

Double Mutation ◽

Specific Amino Acid ◽

Stable Transfectants ◽

Glycosylation Sites ◽

Fviii Activity ◽

A2 Domain

Abstract A major obstacle for gene therapy of hemophilia A is the achievement of adequate factor VIII (fVIII) expression. Bioengineering strategies have targeted specific sequences within human fVIII that are thought to be responsible for its generally poor expression. Specific amino acid substitutions, L303E/F309S herein referred to as double mutation (DM), function to decrease fVIII binding to BiP, a resident ER chaperone, which results in increased fVIII secretion (Swaroop, Moussalli et al. 1997). Furthermore, addition of 6 N-linked glycosylation sites, designated 226/N6, located within the human B domain also increases human fVIII expression (Miao, Sirachainan et al. 2004). We previously demonstrated that porcine and certain hybrid human/porcine fVIII constructs are expressed at 10 – 14-fold greater levels than human fVIII (Doering, Healey et al. 2002; Doering, Healey et al. 2004). The aim of the current study was to directly compare various fVIII expression constructs in order to determine an optimal transgene for gene therapy applications. The following fVIII constructs were generated: human B-domain-deleted fVIII (HBDD-fVIII), HBDD-fVIII with a 14 amino acid linker between the A2 domain and the activation peptide (HSQ-fVIII), porcine fVIII containing a 24 amino acid linker (HEP-fVIII), hybrid human/porcine-fVIII which has porcine A1 and A3 domains (HP47), and modified HBDD, HSQ and HEP-fVIII constructs containing DM and/or 226/N6. Each construct was transiently transfected into BHK-M cells, and fVIII production between 48 – 72 hrs post-transfection was measured using a one-stage clotting assay. Under these conditions, the addition of the DM and 226/N6 significantly increased fVIII expression for HBDD (P = 0.003), though not for HSQ. Addition of DM or 226/N6 alone did not significantly increase the expression of either human fVIII construct, and furthermore, the addition of DM to HEP-fVIII decreased its expression 98%. HEP-fVIII was expressed at 8-fold or greater levels than any of the other human constructs. Next, ~25 stably transfected BHK-M clones were isolated following transfection with each of the fVIII expression constructs and the rate of fVIII production for each clone was determined. Several clones did not express detectable fVIII activity (<0.01 units/mL) and were excluded from the analysis. Approximately 14% of the total number of clones were excluded, ranging from 0 – 42% for the different constructs. HEP-DM-fVIII was the exception, where 82% of the clones had activity <0.01 units/mL. Mean HEP-fVIII expression was 3.93 ± 3.22 units/mL/24 hr (n = 19) (Figure 1), and HP47 was similarly expressed at 3.21 ± 2.31 units/mL/24 hr (n = 19). All of the HSQ-based constructs and HBDD-DM/226/N6 showed similar mean expression levels (0.28 ± 0.03 units/mL/24 hr) and were significantly higher than HBDD and HBDD-DM, which had a mean of 0.12 ± 0.01 units/mL. In the current study, we provide experimental evidence that the expression of HEP-fVIII and HP47 is superior to other bioengineered fVIII expression constructs, which should eliminate the expression barrier that has hampered the clinical translation of gene therapy for hemophilia A. Figure 1: Stable Transfectants Figure 1:. Stable Transfectants

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High-Expression Porcine FVIII Driven by Erythroid-Specific Promoters for Lentiviral Vector-Mediated Gene Therapy

Blood ◽

10.1182/blood.v112.11.3544.3544 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3544-3544

Author(s):

Nadia Sutherland ◽

Kerry L Dooriss ◽

David A McCarty ◽

Christopher B Doering ◽

H. Trent Spencer

Keyword(s):

Gene Therapy ◽

Factor Viii ◽

Blood Cells ◽

Copy Number ◽

Hemophilia A ◽

Lentiviral Vector ◽

K562 Cells ◽

Dna Copy Number ◽

Normal Human ◽

Globin Promoter

Abstract Hemophilia A is an X-linked gene disorder that results in a deficiency of circulating coagulation factor VIII (fVIII) and may be ameliorated by only modest amounts of circulating protein, which makes it a logical candidate for gene therapy. Due to the potential risk of insertional mutagenesis from oncoretroviral-mediated gene therapy, cell-specific expression of transgenes using self-inactivating viral vectors may provide a safer gene therapy approach for use in humans. Therefore, we constructed simian immunodeficiency virus (SIV)-based lentiviral vectors containing a 5′ long-terminal repeat (LTR) and 3′ LTR with self-inactivating U3 deletion, the bovine growth hormone polyA signal, a packaging signal (ψ), and a single internal ankyrin-1 or β-globin promoter, designated SIV-Ank and SIV-Bg, respectively. The minimal 314-bp ankyrin-1 promoter and 180-bp β-globin promoter flanked upstream by enhancing sequences, HS2, HS3, and HS4 (Hanawa et al., Hum Gene Ther, 2002) from the locus control region were cloned into the SIV vector backbone upstream from either enhanced green fluorescent protein (eGFP) or B-domain deleted porcine factor VIII (BDDpfVIII). The erythroid-specificity of each promoter was evaluated in vitro by measurement of either eGFP or fVIII expression following transduction of SIV-Ank and SIV-Bg constructs into both K562 myelogenous leukemic cells and 293T human embryonic kidney cells. GFP expression, as measured by flow cytometry, in transduced cells revealed that the ankyrin-1 and β-globin promoters are more active in K562 cells as compared to 293T cells. The β-globin promoter yielded higher mean fluorescent intensity values for GFP compared to the ankyrin-1 promoter at similar MOIs in K562 cells, suggesting stronger β-globin promoter activity in these cells. Transduction of cells with the SIV vector encoding BDDpfVIII driven by the β-globin promoter resulted in a 14-fold higher number of transcripts per DNA copy number in K562 cells compared to 293T cells, while cells transduced with the ankyrin-l promoter had only a 1.4-fold greater number of transcripts per DNA copy number. In addition, SIV-Bg-fVIII-modified K562 cells produced a 5.2-fold greater number of transcripts per DNA copy number than SIV-Ank- fVIII-modified cells. To evaluate the usefulness of these vectors for in vivo expression of BDDpfVIII, hemophilia A mice (exon 16 knockout) were conditioned with 11 Gy total body irradiation and transplanted with gene-modified Sca-1+ cells transduced with either SIV-Ank-fVIII, SIV-Ank-eGFP, SIV-Bg-fVIII, or SIV-Bg-eGFP. The expression of eGFP from donor red blood cells in recipient mice was approximately 8–12% using both the ankyrin-1 and β-globin promoter constructs. Mice that received cells transduced with SIVAnk- fVIII demonstrated therapeutic levels of plasma fVIII up to 0.5 units/mL (i.e. 50% normal human levels). However, fVIII expression decreased over time and real-time PCR analysis of peripheral blood cells confirmed the loss of detectable fVIII transgene by 6 weeks after transplantation, suggesting there was predominantly gene transfer into short-term repopulating hematopoietic cells. Mice transplanted with SIV-Bg-fVIII-modified hematopoietic stem cells demonstrated a similar rise and fall of fVIII expression within the first 4 weeks after transplantation, and showed an increase in fVIII expression by 6 weeks. At 8 weeks post transplantation, fVIII levels greater than 300% normal human levels were observed. Red blood cell count, hemoglobin, and red blood cell morphology were normal despite the high level of expression of fVIII. Overall these data demonstrate the potential for therapeutic expression of factor VIII using a self-inactivating lentiviral vector containing an erythroid-specific internal promoter.

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Eradication of neutralizing antibodies to factor VIII in canine hemophilia A after liver gene therapy

Blood ◽

10.1182/blood-2010-06-288001 ◽

2010 ◽

Vol 116 (26) ◽

pp. 5842-5848 ◽

Cited By ~ 100

Author(s):

Jonathan D. Finn ◽

Margareth C. Ozelo ◽

Denise E. Sabatino ◽

Helen W. G. Franck ◽

Elizabeth P. Merricks ◽

...

Keyword(s):

Gene Therapy ◽

Factor Viii ◽

Neutralizing Antibodies ◽

Viral Vectors ◽

Hemophilia A ◽

Major Complication ◽

Complete Disappearance ◽

Anamnestic Response ◽

Endogenous Expression ◽

Liver Gene

Abstract Inhibitory antibodies to factor VIII (FVIII) are a major complication in the treatment of hemophilia A, affecting approximately 20% to 30% of patients. Current treatment for inhibitors is based on long-term, daily injections of large amounts of FVIII protein. Liver-directed gene therapy has been used to induce antigen-specific tolerance, but there are no data in hemophilic animals with pre-existing inhibitors. To determine whether sustained endogenous expression of FVIII could eradicate inhibitors, we injected adeno-associated viral vectors encoding canine FVIII (cFVIII) in 2 strains of inhibitor hemophilia A dogs. In 3 dogs, a transient increase in inhibitor titers (up to 7 Bethesda Units [BU]) at 2 weeks was followed by continuous decline to complete disappearance within 4-5 weeks. Subsequently, an increase in cFVIII levels (1.5%-8%), a shortening of clotting times, and a reduction (> 90%) of bleeding episodes were observed. Immune tolerance was confirmed by lack of antibody formation after repeated challenges with cFVIII protein and normal protein half-life. A fourth dog exhibited a strong early anamnestic response (216 BU), with slow decline to 0.8 BU and cFVIII antigen detection by 18 months after vector delivery. These data suggest that liver gene therapy has the potential to eradicate inhibitors and could improve the outcomes of hemophilia A patients.

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Expression of the blood-clotting factor-VIII cDNA is repressed by a transcriptional silencer located in its coding region

Blood ◽

10.1182/blood.v85.9.2447.bloodjournal8592447 ◽

1995 ◽

Vol 85 (9) ◽

pp. 2447-2454 ◽

Cited By ~ 51

Author(s):

RC Hoeben ◽

FJ Fallaux ◽

SJ Cramer ◽

DJ van den Wollenberg ◽

H van Ormondt ◽

...

Keyword(s):

Gene Therapy ◽

Factor Viii ◽

Hemophilia A ◽

Recombinant Dna ◽

Transcription Unit ◽

Current Treatment ◽

Clotting Factor ◽

Large Animal ◽

Coding Region ◽

Large Animal Models

Hemophilia A is caused by a deficiency of factor-VIII procoagulant (fVIII) activity. The current treatment by frequent infusions of plasma-derived fVIII concentrates is very effective but has the risk of transmittance of blood-borne viruses (human immunodeficiency virus [HIV], hepatitis viruses). Use of recombinant DNA-derived fVIII as well as gene therapy could make hemophilia treatment independent of blood-derived products. So far, the problematic production of the fVIII protein and the low titers of the fVIII retrovirus stocks have prevented preclinical trials of gene therapy for hemophilia A in large-animal models. We have initiated a study of the mechanisms that oppose efficient fVIII synthesis. We have established that fVIII cDNA contains sequences that dominantly inhibit its own expression from retroviral as well as from plasmid vectors. The inhibition is not caused by instability of the fVIII mRNA (t1/2, > or = 6 hours) but rather to repression at the level of transcription. A 305-bp fragment is identified that is involved in but not sufficient for repression. This fragment does not overlap the region recently identified by Lynch et al (Hum Gene Ther 4:259, 1993) as a dominant inhibitor of RNA accumulation. The repression is mediated by a cellular factor (or factors) and is independent of the orientation of the element in the transcription unit, giving the repressor element the hallmarks of a transcriptional silencer.

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Gene therapy in hemophilia A: a cost-effectiveness analysis

Blood Advances ◽

10.1182/bloodadvances.2018021345 ◽

2018 ◽

Vol 2 (14) ◽

pp. 1792-1798 ◽

Cited By ~ 12

Author(s):

Nicoletta Machin ◽

Margaret V. Ragni ◽

Kenneth J. Smith

Keyword(s):

Gene Therapy ◽

Cost Effectiveness ◽

Factor Viii ◽

Time Horizon ◽

Hemophilia A ◽

Cost Effective ◽

Severe Hemophilia ◽

Effectiveness Analysis ◽

Key Points ◽

Therapy Cost

Key Points Gene therapy is cost-effective in severe hemophilia A compared with standard factor VIII prophylaxis. Over a 10-year time horizon, gene therapy cost $1M and resulted in 8.33 QALYs gained, whereas prophylaxis cost $1.7M and resulted in 6.62 QALYs gained.

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Development And Application Of A Minimal-Adenoviral Vector System For Gene Therapy Of Hemophilia A

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615881 ◽

1999 ◽

Vol 82 (08) ◽

pp. 562-571 ◽

Cited By ~ 14

Author(s):

Steven Josephs ◽

Jiemin Zhou ◽

Xiangming Fang ◽

Ramón Alemany ◽

Cristina Balagué ◽

...

Keyword(s):

Gene Therapy ◽

Factor Viii ◽

Human Factor ◽

Hemophilia A ◽

Vector System ◽

High Level Expression ◽

Viral Particles ◽

Human Factor Viii ◽

High Level

IntroductionHemophilia A and B are the most common bleeding disorders caused by deficiencies of clotting factors VIII and IX, respectively, both of which are X-linked with a recessive heredity.1 Replacement of the deficient factors with frequent intravenous injections of plasma concentrates or recombinant proteins is the standard treatment for these diseases.2 Great efforts have been made for nearly a decade toward developing experimental gene therapy for these diseases and aiming at the development of a medical intervention that is more effective and convenient than the currently available replacement therapies.3 Hemophilia is a suitable clinical model for the development of gene therapy products and has a number of advantages: 1) there is a simple and well defined cause-and-effect relationship between the protein deficiencies and bleeding symptoms; 2) tissue-specific expression and precise regulation of the transgenes are not necessary; 3) well characterized animal models are available for preclinical studies; 4) an unequivocal endpoint for product efficacy can be assessed in clinical trials; and 5) even 1% to 5% of the normal physiological levels of the proteins is therapeutic.For gene therapy of hemophilia, the most challenging hurdle, with respect to the long-term expression of the deficient proteins at adequate levels, is the development of a suitable gene delivery system. Technologies have been evolving from ex vivo to in vivo approaches, from initial use of retroviral vector to recent application of adenviral (Ad) or adeno-associated virus (AAV) vector, demonstrating progress from early results of transient low-level expression to more sustained high-level expression.3 For hemophilia A treatment, Ad vectors are particularly useful, since the liver naturally produces factor VIII, and following intravenous (i.v.) injection, Ad vectors concentrate in the liver. This makes the gene transduction efficiency to liver very high. Adenovirus vectors have been developed for gene therapy due to their high titer, broad infectivity, potential for large payload, and in vivo gene delivery capacity.4 Although the immunogenicity and cytotoxicity associated with the early-generation Ad vectors have been a concern with respect to their clinical application, newly developed vectors, in which the viral coding sequences have been deleted, have significantly reduced the side effects associated with the vectors. The “gutless” Ad vector, or so called helper-dependent, large-capacity, or mini- Ad vectors are the representative examples of these new-generation Ad vectors.5-15 The mini-Ad vector system described in this report was developed based on two major research findings. First, an Ad- SV40 hybrid virus discovered during attempts to grow human Ad in non-permissive monkey COS-7 cells.16 The hybrid virus had a genome structure in which only both ends of the Ad sequences were retained and almost all coding sequences of the Ad genome were replaced by symmetric, tandemly repeated SV40 genomes. The hybrid viruses replicated and were packaged in the presence of a wild-type Ad as a helper. This finding implied that total replacement of the Ad genome was possible to form a mini-Ad vector as long as proper helper function and selective pressure was provided. Secondly, it was discovered that Ad packaging can be attenuated by deleting portions of the packaging signal.17 This finding provided a means to put selective pressure on the helper Ad (referred to as ancillary Ad) by specifically limiting its packaging process and allowing a preferential packaging of the mini-Ad. The system, therefore, is designed to have three main components: the mini-Ad vector, the E1-deleted ancillary Ad, and a production cell line that provides AdE1 complementation.Based on the mini-Ad vector system, MiniAdFVIII was developed. The MiniAdFVIII vector carries a 27 kb expression cassette, in which the full-length human factor VIII cDNA is flanked by a human albumin promoter and cognate genomic sequences. Infection of MiniAdFVIII in vitro showed that the vector mediated expression of functional human factor VIII at levels of 100-200 ng/106 cells per 24 hours in HepG2 and 293 cells. With single-dose intravenous injection of 1011 viral particles in hemophilic mice, MiniAdFVIII produced a sustained high-level expression of human factor VIII (at 100-800 ng/ml for up to 369 days) that corrected the factor VIII-deficient phenotype. Safety studies of MiniAdFVIII showed that there were no significant toxicities in mice and dogs after a single intravenous dose of up to 3×1011 and 6×1012 viral particles, respectively. In this report, other studies for developing the MiniAdFVIII vector with a site-specific integration capability and the development of a human factor VIII-tolerized mouse model for preclinical studies of MiniAdFVIII are described.

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