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      Bioengineered coagulation factor VIII enables long-term correction of murine hemophilia A following liver-directed adeno-associated viral vector delivery

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          Abstract

          Clinical data support the feasibility and safety of adeno-associated viral (AAV) vectors in gene therapy applications. Despite several clinical trials of AAV-based gene transfer for hemophilia B, a unique set of obstacles impede the development of a similar approach for hemophilia A. These include (i) the size of the factor VIII (fVIII) transgene, (ii) humoral immune responses to fVIII, (iii) inefficient biosynthesis of human fVIII, and (iv) AAV vector immunity. Through bioengineering approaches, a novel fVIII molecule, designated ET3, was developed and shown to improve biosynthetic efficiency 10- to 100-fold. In this study, the utility of ET3 was assessed in the context of liver-directed, AAV-mediated gene transfer into hemophilia A mice. Due to the large size of the expression cassette, AAV-ET3 genomes packaged into viral particles as partial genome fragments. Despite this potential limitation, a single peripheral vein administration of AAV-ET3 into immune-competent hemophilia A mice resulted in correction of the fVIII deficiency at lower vector doses than previously reported for similarly oversized AAV-fVIII vectors. Therefore, ET3 appears to improve vector potency and mitigate at least one of the critical barriers to AAV-based clinical gene therapy for hemophilia A.

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          Quantitative analysis of the packaging capacity of recombinant adeno-associated virus.

          J. Dong, P Fan, Margaret Frizzell (1996)
          Recombinant adeno-associated viruses (AAV) are among the most promising vectors for gene therapy of genetic diseases, including cystic fibrosis (CF). However, because of its small genome size, the capacity of AAV to package a therapeutic gene is limited. The efficiency of packaging the cystic fibrosis transmembrane conductance Regulator (CFTR) gene into AAV will be an important factor in determining whether recombinant AAV can be developed as a vector for transferring CFTR cDNA to the airway epithelia of patients with CF. Current understanding of the AAV biology suggests that AAV can package a genome slightly larger than the size of a wild-type genome. The precise range of the genome size and the efficiency of packaging have not been defined. Using a series of AAV vectors with progressively-increasing genome size, we were able to analyze quantitatively the packaging efficiency in relation to the vector size and to determine the size limit for packaging. The packaging efficiencies of AAV vectors of variable sizes were determined directly by assaying DNA contents of viral particles, and indirectly by analyzing their efficiency in transfer of a chloramphenicol acetyltransferase (CAT) reporter gene into target cells. Our studies showed that the optimal size of AAV vector is between 4.1 and 4.9 kb. Although AAV can package a vector larger than its genome size, up to 5.2 kb, the packaging efficiencies in this large size range were sharply reduced. When the AAV genome size was smaller than 4.1 kb, the packaging efficiency was also suboptimal. In contrast, when the size of the genome was less than half the length of the wild-type genome, two copies of the vector were packaged into each virion, suggesting that the copy number control during packaging is a "head-full" mechanism. Because the length of the minimal cDNA of CFTR is about 4.5 kb, these results suggest it is possible to package the CFTR gene into AAV if the combined length of transcriptional elements and ITRs is kept under 500 bp. The results of this study are important for directing the design of AAV vectors for efficient gene transfer, as well as for a better understanding of the mechanism of AAV genome packaging.
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            Packaging capacity of adeno-associated virus serotypes: impact of larger genomes on infectivity and postentry steps.

            Richard J. Samulski, Joshua Grieger (2005)
            The limited packaging capacity of adeno-associated virus (AAV) precludes the design of vectors for the treatment of diseases associated with larger genes. Autonomous parvoviruses, such as minute virus of mice and B19, while identical in size (25 nm), are known to package larger genomes of 5.1 and 5.6 kb, respectively, compared to AAV genomes of 4.7 kb. One primary difference is the fact that wild-type (wt) AAV utilizes three capsid subunits instead of two to form the virion shell. In this study, we have characterized the packaging capacity of AAV serotypes 1 through 5 with and without the Vp2 subunit. Using reporter transgene cassettes that range in size from 4.4 to 6.0 kb, we determined that serotypes 1 through 5 with and without Vp2 could successfully package, replicate in, and transduce cells. Dot blot analysis established that packaging efficiency was similar for all vector cassettes and that the integrity of encapsidated genomes was intact regardless of size. Although physical characterization determined that virion structures were indistinguishable from wt, transduction experiments determined that all serotype vectors carrying larger genomes (5.3 kb and higher) transduced cells less efficiently (within a log) than AAV encapsidating wt size genomes. This result was not unique to reporter genes and was observed for CFTR vector cassettes ranging in size from 5.1 to 5.9 kb. No apparent advantage in packaging efficiency was observed when Vp2 was present or absent from the virion. Further analysis determined that a postentry step was responsible for the block in infection and specific treatment of cells upon infection with proteasome inhibitors increased transduction of AAV encapsidating larger DNA templates to wt levels, suggesting a preferential degradation of virions encapsidating larger-than-wt genomes. This study illustrates that AAV is capable of packaging and protecting recombinant genomes as large as 6.0 kb but the larger genome-containing virions are preferentially degraded by the proteasome and that this block can be overcome by the addition of proteasome inhibitors.
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              Therapeutic levels of FVIII following a single peripheral vein administration of rAAV vector encoding a novel human factor VIII variant.

              S. Rabbanian, Jenny McIntosh, Andrew Davidoff … (2013)
              Recombinant adeno-associated virus (rAAV) vectors encoding human factor VIII (hFVIII) were systematically evaluated for hemophilia A (HA) gene therapy. A 5.7-kb rAAV-expression 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 ± 162% of normal) in HA knock-out mice following administration of 2 × 10(12) vector genomes/kg, a vector dose shown to be safe in subjects with hemophilia B. Stable hFVIII expression at 15 ± 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.
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                Author and article information

                Journal
                Journal ID (nlm-ta): Mol Ther Methods Clin Dev
                Journal ID (iso-abbrev): Mol Ther Methods Clin Dev
                Title: Molecular Therapy. Methods & Clinical Development
                Publisher: Nature Publishing Group
                ISSN (Electronic): 2329-0501
                Publication date (Electronic): 06 August 2014
                Publication date Collection: 2014
                Volume: 1
                Page: 14036
                Affiliations
                [1 ]Graduate Program in Molecular and Systems Pharmacology, Laney Graduate School, Emory University , Atlanta, Georgia, USA
                [2 ]Center for Cellular and Molecular Therapeutics, The Children’s Hospital of Philadelphia , Philadelphia, Pennsylvania, USA
                [3 ]Perelman School of Medicine, University of Pennsylvania , Philadelphia, Pennsylvania, USA
                [4 ]Department of Pediatrics, Aflac Cancer and Blood Disorders Center, Emory University School of Medicine , Atlanta, Georgia, USA
                Author notes
                Article
                Publisher Item ID: mtm201436
                DOI: 10.1038/mtm.2014.36
                PMC ID: 4362354
                PubMed ID: 26015976
                SO-VID: 3a6fb501-7321-4c4f-8b9f-38daf1696598
                Copyright © Copyright © 2014 American Society of Gene & Cell Therapy
                License:

                This work is licensed under a Creative Commons Attribution 3.0 Unported License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/3.0/

                History
                Date received : 05 December 2013
                Date revision received : 04 June 2014
                Date accepted : 04 June 2014
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