The Enhancing Effects of the Light Chain on Heavy Chain Secretion in Split Delivery of Factor VIII Gene

Chen, Lingxia; Zhu, Faming; Li, Juan; Lu, Hui; Jiang, Haiyan; Sarkar, Rita; Arruda, Valder R.; Wang, Jinhui; Zhao, Jennifer; Pierce, Glenn F.; Ding, Qiulan; Wang, Xuefeng; Wang, Hongli; Pipe, Steven W.; Liu, Xiang Qin; Xiao, Xiao; Camire, Rodney M.; Xiao, Weidong

doi:10.1038/sj.mt.6300268

Cited by 40 publications

(55 citation statements)

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“…13 Other innovative approaches to overcome the size constraint involve the need for simultaneous transduction with 2 AAV vectors for functional FVIII activity, which has limited their transition to the clinic. [14][15][16][17] We and others have shown that expression of FVIII can be improved in the context of lentiviral vectors by reorganization of the wildtype cDNA of hFVIII according to the codon usage of highly expressed human genes as described before for human factor IX (hFIX). 3,18,19 However, these codon-optimized (codop) FVIII variants remain hitherto untested in the setting of recombinant AAV (rAAV).…”

mentioning

confidence: 99%

Therapeutic levels of FVIII following a single peripheral vein administration of rAAV vector encoding a novel human factor VIII variant

McIntosh

Lenting

Rosales

et al. 2013

Blood

235

267

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• Novel, more potent codonoptimized human FVIII variant (codop-hFVIII-V3).• Codop-hFVIII-V3 is safe and efficacious in mice and nonhuman primates, thus improving the prospects of gene therapy for hemophilia A.Recombinant adeno-associated virus (rAAV) vectors encoding human factor VIII (hFVIII) were systematically evaluated for hemophilia A (HA) gene therapy. A 5.7-kb rAAVexpression cassette (rAAV-HLP-codop-hFVIII-N6) containing a codon-optimized hFVIII cDNA in which a 226 amino acid (aa) B-domain spacer replaced the entire B domain and a hybrid liver-specific promoter (HLP) mediated 10-fold higher hFVIII levels in mice compared with non-codon-optimized variants. A further twofold improvement in potency was achieved by replacing the 226-aa N6 spacer with a novel 17-aa peptide (V3) in which 6 glycosylation triplets from the B domain were juxtaposed. The resulting 5.2-kb rAAV-HLP-codop-hFVIII-V3 cassette was more efficiently packaged within AAV virions and mediated supraphysiologic hFVIII expression (732 6 162% of normal) in HA knockout mice following administration of 2 3 10 12 vector genomes/kg, a vector dose shown to be safe in subjects with hemophilia B. Stable hFVIII expression at 15 6 4% of normal was observed at this dose in a nonhuman primate. hFVIII expression above 100% was observed in 3 macaques that received a higher dose of either this vector or the N6 variant. These animals developed neutralizing anti-FVIII antibodies that were abrogated with transient immunosuppression. Therefore, rAAV-HLP-codop-hFVIII-V3 substantially improves the prospects of effective HA gene therapy. (Blood. 2013;121(17):3335-3344) Introduction Hemophilia A (HA), the most common inherited bleeding disorder, caused by a deficiency of factor VIII (FVIII) is well suited for a gene replacement approach, primarily because a modest increase in the level of FVIII (.1% of normal) can ameliorate the severe bleeding phenotype.1 Several gene transfer strategies for FVIII replacement have been evaluated. 2 However, adeno-associated viral (AAV) vectors currently show the greatest promise because of their excellent safety profile and ability to direct long-term transgene expression from postmitotic tissues such as the liver.3-5 Indeed, our ongoing gene therapy clinical trial for hemophilia B, a related bleeding diathesis, has demonstrated that a single peripheral vein administration of AAV vector leads to stable (.30 months) expression of human factor IX (FIX) at levels between 1% to 6% of normal. This is sufficient for conversion of the hemophilia phenotype from severe to moderate or mild.5 Two-thirds of the participants in this study have discontinued prophylaxis and remain free of spontaneous hemorrhage. The other participants have increased the interval between FIX prophylaxes. The use of AAV vectors for HA gene therapy, however, poses new challenges because of the distinct molecular and biochemical properties of human FVIII (hFVIII). Compared with other proteins of similar size, expression of hFVIII is highly inefficient.6 Bioengineeri...

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confidence: 99%

Therapeutic levels of FVIII following a single peripheral vein administration of rAAV vector encoding a novel human factor VIII variant

McIntosh

Lenting

Rosales

et al. 2013

Blood

235

267

View full text Add to dashboard Cite

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“…In addition, N-linked oligosaccharides in the B-domain of the FVIII heavy chain can also facilitate FVIII secretion by participating in the folding interactions within the ER as well as potentially helping facilitate ER-Golgi transport [16]. Our previous work has shown that dual-vector BDD-FVIII gene transfected cells have similar expression levels of intracellular HC and LC [10]. Therefore, dual-vector mediated FVIII gene transfer can overcome the capacity constraints of AAV vectors, but the inefficient secretion of the HC leads to its excessive intracellu-lar deposition, which not only reduces the efficacy of gene therapy, but may also destabilize host cells and possibly induce apoptosis [17].…”

Section: Discussionmentioning

confidence: 99%

“…When an inter-chain disulfide bond is formed, the secretion of HC increased likely driven by covalently bonded LC. In our recent studies using protein trans-splicing mediated dual-vector FVIII gene co-transfection in cultured cells, a cis-promoting effect of LC covalently linked to the HC by peptide bond on the HC secretion was found [6,10].…”

Section: Discussionmentioning

confidence: 99%

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Enhanced plasma factor VIII activity in mice via cysteine mutation using dual vectors

Zhu

Liu

Jiang

et al. 2012

Sci. China Life Sci.

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Hemophilia A is caused by a genetic mutation in coagulation factor VIII (FVIII) gene and gene therapy is considered to be a promising strategy for its treatment. We recently demonstrated that co-delivery of two vectors expressing M662C mutated heavy and D1828C mutated light chain genes of B-domain-deleted coagulation factor VIII (BDD-FVIII) leads to inter-chain disulfide cross-linking and improved heavy chain secretion in vitro. In this study, co-injection of both M662C and D1828C mutated BDD-FVIII gene expression vectors into mice resulted in increased heavy chain secretion and coagulation activity in plasma in vivo. Approximately (239±56) ng mL 1 above endogenous levels of transgenic FVIII heavy chain was found in mouse plasma using a chain-specific ELISA. For FVIII coagulation activity, approximately (1.09±0.25) IU mL 1 above endogenous levels were detected in co-injected transgenic mouse plasma using a chromogenic assay. These data demonstrate that inter-chain disulfide bonds likely increase heavy chain secretion and coagulation activity in the plasma of transgenic mice with an improved efficacy of a dual-vector delivery of BDD-FVIII gene. These findings support our ongoing efforts to develop a gene therapy for hemophilia A treatment using dual-AAV vectors.coagulation factor VIII, dual-vector gene delivery, inter-chain disulfide bonding, heavy chain secretion, coagulation activity Citation: Zhu F X, Liu Z L, Miao J, et al. Enhanced plasma factor VIII activity in mice via cysteine mutation using dual vectors. Sci China Life Sci, 2012, 55: 521 -526,

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“…[168] Because of its simplicity, high efficiency and reproducibility, hydrodynamic gene delivery has been utilized for the delivery of DNA, small interfering RNA, proteins, small compounds and even viral vectors. [171][172][173][174][175][176][177][178][179] This technique has been widely used for gene therapy studies, gene knockdown, functional analysis of genetic elements and for establishing a disease model in animals. [171,176,180] In an attempt to apply this simple procedure of gene delivery to the clinic, efforts have been made to reduce the total injection volume.…”

Section: Hydrodynamic Gene Delivery (Hydroporation)mentioning

confidence: 99%

Advances in Gene Delivery Systems

et al. 2011

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The transfer of genes into cells, both in vitro and in vivo, is critical for studying gene function and conducting gene therapy. Methods that utilize viral and nonviral vectors, as well as physical approaches, have been explored. Viral vector-mediated gene transfer employs replication-deficient viruses such as retro-virus, adenovirus, adeno-associated virus and herpes simplex virus. A major advantage of viral vectors is their high gene delivery efficiency. The nonviral vectors developed so far include cationic liposomes, cationic polymers, synthetic peptides and naturally occurring compounds. These nonviral vectors appear to be highly effective in gene delivery to cultured cells in vitro but are significantly less effective in vivo. Physical methods utilize mechanical pressure, electric shock or hydrodynamic force to transiently permeate the cell membrane to transfer DNA into target cells. They are simpler than viral-and nonviral-based systems and highly effective for localized gene delivery. The past decade has seen significant efforts to establish the most desirable method for safe, effective and target-specific gene delivery, and good progress has been made. The objectives of this review are to (i) explain the rationale for the design of viral, nonviral and physical methods for gene delivery; (ii) provide a summary on recent advances in gene transfer technology; (iii) discuss advantages and disadvantages of each of the most commonly used gene delivery methods; and (iv) provide future perspectives.

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The Enhancing Effects of the Light Chain on Heavy Chain Secretion in Split Delivery of Factor VIII Gene

Cited by 40 publications

References 34 publications

Therapeutic levels of FVIII following a single peripheral vein administration of rAAV vector encoding a novel human factor VIII variant

Therapeutic levels of FVIII following a single peripheral vein administration of rAAV vector encoding a novel human factor VIII variant

Enhanced plasma factor VIII activity in mice via cysteine mutation using dual vectors

Advances in Gene Delivery Systems

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