Direct Gene Replacement Research Articles

FVIII expression by its native promoter sustains long-term correction avoiding immune response in hemophilic mice

AbstractHere we describe a successful gene therapy approach for hemophilia A (HA), using the natural F8 promoter (pF8) to direct gene replacement to factor VIII (FVIII)–secreting cells. The promoter sequence and the regulatory elements involved in the modulation of F8 expression are still poorly characterized and biased by the historical assumption that FVIII expression is mainly in hepatocytes. Bioinformatic analyses have highlighted an underestimated complexity in gene expression at this locus, suggesting an activation of pF8 in more cell types than those previously expected. C57Bl/6 mice injected with a lentiviral vector expressing green fluorescent protein (GFP) under the pF8 (lentiviral vector [LV].pF8.GFP) confirm the predominant GFP expression in liver sinusoidal endothelial cells, with a few positive cells detectable also in hematopoietic organs. Therapeutic gene delivery (LV.pF8.FVIII) in hemophilic C57/Bl6 and 129-Bl6 mice successfully corrected the bleeding phenotype, rescuing up to 25% FVIII activity, using a codon-optimized FVIII, with sustained activity for the duration of the experiment (1 year) without inhibitor formation. Of note, LV.pF8.FVIII delivery in FVIII-immunized HA mice resulted in the complete reversion of the inhibitor titer with the recovery of therapeutic FVIII activity. Depletion of regulatory T cells (Tregs) in LV-treated mice allowed the formation of anti-FVIII antibodies, indicating a role for Tregs in immune tolerance induction. The significant blood loss reduction observed in all LV.pF8.FVIII-treated mice 1 year after injection confirmed the achievement of a long-term phenotypic correction. Altogether, our results highlight the potency of pF8-driven transgene expression to correct the bleeding phenotype in HA, as well as potentially in other diseases in which an endothelial-specific expression is required.

Regulatory and Scientific Advancements in Gene Therapy: State-of-the-Art of Clinical Applications and of the Supporting European Regulatory Framework.

Advanced therapy medicinal products (ATMPs) have a massive potential to address existing unmet medical needs. Specifically, gene therapy medicinal products (GTMPs) may potentially provide cure for several genetic diseases. In Europe, the ATMP regulation was fully implemented in 2009 and, at this point, the Committee for Advanced Therapies was created as a dedicated group of specialists to evaluate medicinal products requiring specific expertise in this area. To date, there are three authorized GTMPs, and the first one was approved in 2012. Broad research has been conducted in this field over the last few decades and different clinical applications are being investigated worldwide, using different strategies that range from direct gene replacement or addition to more complex pathways such as specific gene editing or RNA targeting. Important safety risks, limited efficacy, manufacturing hurdles, or ethical conflicts may represent challenges in the success of a candidate GTMP. During the development process, it is fundamental to take such aspects into account and establish overcoming strategies. This article reviews the current European legal framework of ATMPs, provides an overview of the clinical applications for approved and investigational GTMPs, and discusses critical challenges in the development of GTMPs.

Gene Replacement Therapies for Duchenne Muscular Dystrophy Using Adeno-Associated Viral Vectors

The muscular dystrophies collectively represent a major health challenge, as few significant treatment options currently exist for any of these disorders. Recent years have witnessed a proliferation of novel approaches to therapy, spanning increased testing of existing and new pharmaceuticals, DNA delivery (both anti-sense oligonucleotides and plasmid DNA), gene therapies and stem cell technologies. While none of these has reached the point of being used in clinical practice, all show promise for being able to impact different types of muscular dystrophies. Our group has focused on developing direct gene replacement strategies to treat recessively inherited forms of muscular dystrophy, particularly Duchenne and Becker muscular dystrophy (DMD/BMD). Both forms of dystrophy are caused by mutations in the dystrophin gene and all cases can in theory be treated by gene replacement using synthetic forms of the dystrophin gene. The major challenges for success of this approach are the development of a suitable gene delivery shuttle, generating a suitable gene expression cassette able to be carried by such a shuttle, and achieving safe and effective delivery without elicitation of a destructive immune response. This review summarizes the current state of the art in terms of using adeno-associated viral vectors to deliver synthetic dystrophin genes for the purpose of developing gene therapy for DMD.

Knocking out the bad allele.

Knocking out the bad allele.

An efficient method for precise gene substitution in the AcMNPV genome by homologous recombination in E. coli

The RecA-mediated homologous recombination method was improved and used to direct gene replacement in baculoviruses. With this method, the p74 gene in the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) genome was substituted precisely by the p74 gene of Spodoptera litura multicapsid nucleopolyhedrovirus (SpltMNPV). In the recombinant bacmid, the AcMNPV p74 gene promoter controlled directly the expression of SpltMNPV p74 gene. Results of RT-PCR showed transcription of SpltMNPV p74 gene in the recombinant, implying the potential use of this easy and efficient method in baculovirus gene function research.

A Genetic Screen for Aminophospholipid Transport Mutants Identifies the Phosphatidylinositol 4-Kinase, Stt4p, as an Essential Component in Phosphatidylserine Metabolism

In an effort to understand molecular mechanisms of intracellular lipid transport, we have focused upon specific events required for de novo aminophospholipid synthesis in the yeast Saccharomyces cerevisiae. A genetic system for examining the steps between phosphatidylserine (PtdSer) synthesis in the endoplasmic reticulum and its transport to and decarboxylation by PtdSer decarboxylase 2 in the Golgi/vacuole has been developed. We have isolated a mutant, denoted pstB1, that accumulates PtdSer and has diminished phosphatidylethanolamine formation despite normal PtdSer decarboxylase 2 activity. The lesion in PtdSer metabolism is consistent with a defect in interorganelle lipid transport. A genomic DNA clone that complements the mutation was isolated, and sequencing revealed that the clone contains the STT4 gene, encoding a phosphatidylinositol 4-kinase. The pstB1 mutant exhibits a defect in Stt4p-type phosphatidylinositol 4-kinase activity, and direct gene replacement indicates that STT4 is the defective gene in the mutant. Creation of an STT4 null allele (stt4Delta::HIS3) demonstrates the gene is essential. These results provide evidence that implicates phosphoinositides in the regulation of intracellular aminophospholipid transport.

Direct gene replacement of the mouse α(1,3)‐galactosyltransferase gene with human α(1,2)‐fucosyltransferase gene: Converting α‐galactosyl epitopes into H antigens

Abstract: The chronic donor organ shortage has led to the production of transgenic animals. We assume that cells or organs derived from possible animal donors carrying a large amount of α‐galactosyl epitopes should not be transplanted into humans, because a corresponding amount of immunosuppressants would be needed to prolong the survival of such xenografts in the recipients. This may not only make the recipients compromised hosts but also introduce some unknown or uncontrollable pathogens into society at large. We also assume that gene manipulation itself should not be a detriment to possible transgenic animals. To explore possibilities that not only can minimize the possible detrimental factors to humans, such as α‐galactosyl epitopes, but also can minimize the possible detriment to transgenic animals, such as random integration of the extraneous genes with or without uncontrollable regulatory sequences, we have produced a DNA construct that replaces the mouse oc(1,3)‐galactosyltransferase gene (GT) with the human a(1,2)‐fucosyltransferase (FT) minigene (promoterless for the expression of FT) at the GT locus. The mouse fibrosarcoma cell line, L929, was transfected with the construct. Colonies were obtained after incubation with non‐heat‐inactivated human serum. Southern blot analysis demonstrated that one allele of the mouse GT gene was replaced with the FT minigene at the GT locus without integration of any selectable marker genes. The immunostaining analysis with lectins showed that the transfectants expressed H antigens, which suggested that H antigens were expressed by the intrinsic GT promoter. Thus gene replacement, knock‐in, of the mouse GT with the human FT without integration of any selectable marker genes in the GT locus was shown to be possible. This is especially important in producing transgenic animals for the clinical application of xenografts into humans.

Genetic and molecular characterization of murine GATA-1 in Aspergillus defines a critical role for the N-terminal finger.

We have utilized Aspergillus nidulans as a model system for the characterization of the major vertebrate transcription factor GATA-1. This has been achieved both by analysing the function of murine GATA-1 directly and by using direct gene replacement to introduce chimaeric areA::GATA-1 derivatives at the areA locus, which encodes a GATA factor involved in regulating nitrogen metabolism in A. nidulans. Although GATA-1 shows only limited function when expressed in A. nidulans, the C-terminal GATA DNA-binding domain can replace the native GATA domain of AREA and retain near wild-type function. Surprisingly, inclusion of the N-terminal DNA-binding domain of GATA-1 has a major role in determining the function of areA::GATA constructs in vivo, leading to a general loss of activation. This negative function is partially dominant and is dependent on both the fidelity of the zinc-chelating structure and a second factor encoded by A. nidulans. The presence of two GATA domains also disrupts modulation of AREA activity. The ability of duplicate GATA domains to disrupt normal signal transduction is not dependent on the relative position of the domains or on the fidelity of the zinc-chelating structure. This demonstrates the utility of nitrogen metabolism's regulation in A. nidulans as a model system for the molecular and genetic characterization of heterologous GATA factors while also providing insights into native Aspergillus regulatory components.

Mutational analysis of the C-terminal region of AREA, the transcription factor mediating nitrogen metabolite repression in Aspergillus nidulans.

In Aspergillus nidulans the positive-acting, wide domain regulatory gene areA mediates nitrogen metabolite repression. Previous analysis demonstrated that the C-terminal 153 residues of the areA product (AREA) are inessential for at least partial expression of most genes subject to regulation by areA. Paradoxically, areAr2, a -1 frameshift replacing the wild-type 122 C-terminal residues with a mutant peptide of 117 amino acids, leads to general loss of function. To determine the basis for the areAr2 mutant phenotype, and as a means of delineating functional domains within the C-terminal region of AREA, we have selected and characterised areAr2 revertants. Deletion analysis, utilising direct gene replacement, extended this analysis. A mutant areA product truncated immediately after the last residue of the highly conserved GATA (DNA-binding) domain retains partial function. The areAr2 product retains some function with respect to the expression of uaZ (encoding urate oxidase) and the mutant allele is partially dominant with respect to nitrate reductase levels. Consistent with the areAr2 product having a debilitating biological activity, we have demonstrated that a polypeptide containing both the wild-type DNA-binding domain and the mutant C-terminus of AREA2 is able to bind DNA in vitro but no longer shows specificity for GATA sequences.