Recombinant AAV vectors have been used in clinical trials since the mid-1990s, with over 300 subjects enrolled in studies. Although there are not yet licensed AAV products, there are several clear examples of clinical efficacy, and recombinant AAV vectors have a strong safety record after administration both locally and systemically. This chapter provides a review of two types of studies that have shown efficacy, including studies for Leber's congenital amaurosis, a hereditary retinal degenerative disorder in which subretinal administration of AAV has shown efficacy in terms of improvement in multiple measures of visual/retinal function; and of Parkinson's disease which has also shown improvement in clinical and imaging studies after gene transfer to the CNS. The chapter also provides a detailed review of the results of studies of gene therapy for hemophilia, in which short-term efficacy was achieved, but expression of the donated gene failed to persist, likely due to an immune response to the vector. Safety issues relating to AAV-mediated gene transfer are discussed, including a detailed review of the single death to have occurred in an AAV gene therapy trial (likely unrelated to the AAV vector), and of issues related to integration and insertional mutagenesis, risk of germline transmission, and risks related to immune responses to either vector or transgene product. Finally, protocols for determining the presence of vector DNA in body fluids using real-time quantitative PCR, and for isolating, cryopreserving, and testing peripheral blood mononuclear cells for interferon-γ (IFN-γ) responses to capsid are described in detail.
"Limiting transgene expression to neurons rather than glial cells may be more effective at interrupting intrinsic neurodegenerative processes. The majority of preclinical and clinical gene transfer studies in the CNS have utilized AAV-based vectors, which have an excellent immunogenicity profile and established clinical safety [11,12,13,14,15,16]. However, there are several limitations of the use of AAV. "
[Show abstract][Hide abstract] ABSTRACT: Gene-based therapies for neurological diseases continue to develop briskly. As disease mechanisms are elucidated, flexible gene delivery platforms incorporating transcriptional regulatory elements, therapeutic genes and targeted delivery are required for the safety and efficacy of these approaches. Adenovirus serotype 5 (Ad5)-based vectors can carry large genetic payloads to provide this flexibility, but do not transduce neuronal cells efficiently. To address this, we have developed a tropism-modified Ad5 vector with neuron-selective targeting properties for evaluation in models of Parkinson disease therapy. A panel of tropism-modified Ad5 vectors was screened for enhanced gene delivery in a neuroblastoma cell line model system. We used these observations to design and construct an unbiased Ad vector platform, consisting of an unmodified Ad5 and a tropism-modified Ad5 vector containing the fiber knob domain from canine Ad serotype 2 (Ad5-CGW-CK2). Delivery to the substantia nigra or striatum showed that this vector produced a neuronally-restricted pattern of gene expression. Many of the transduced neurons were from regions with afferent projections to the injection site, implicating that OPEN ACCESS Viruses 2014, 6 3294 the vector binds the presynaptic terminal resulting in presynaptic transduction. We show that Ad5-CGW-CK2 can selectively transduce neurons in the brain and hypothesize that this modular platform is potentially adaptable to clinical use.
"Similar considerations might apply to a novel GT approach based on direct CSF administration of AAV9 that has recently been reported as highly effective in treating the central and somatic pathology of mucopolysaccaridosis IIIA mice (53). Indeed, low but definite risk of integration and insertional mutagenesis, risk of germline transmission and risks related to immune responses to either vector or transgene product are aggravated with the vector dose and its multi-organ diffusion (54). "
[Show abstract][Hide abstract] ABSTRACT: Globoid Cell Leukodystrophy (GLD) is an inherited lysosomal storage disease caused by β-galactocerebrosidase (GALC) deficiency. Gene therapy (GT) should provide rapid, extensive and lifetime GALC supply in CNS tissues to prevent or halt irreversible neurologic progression. Here we used a lentiviral vector (LV) to transfer a functional GALC gene in the brain of Twitcher mice, a severe GLD model. A single injection of LV.GALC in the external capsule of Twitcher neonates resulted in robust transduction of neural cells with minimal and transient activation of inflammatory and immune response. Importantly, we documented a proficient transduction of proliferating and post-mitotic oligodendroglia, a relevant target cell type in GLD. GALC activity (30-50% of physiological levels) was restored in the whole CNS of treated mice as early as 8 days post-injection. The early and stable enzymatic supply ensured partial clearance of storage and reduction of psychosine levels, translating in amelioration of histopathology and enhanced lifespan. At 6 months post-injection in non-affected mice, LV genome persisted exclusively in the injected region, where transduced cells overexpressed GALC. Integration site analysis in transduced brain tissues showed no aberrant clonal expansion and preferential targeting of neural-specific genes. This study establishes neonatal LV-mediated intracerebral GT as a rapid, effective, and safe therapeutic intervention to correct CNS pathology in GLD and provides a strong rationale for its application in this and similar leukodystrophies, alone or in combination with therapies targeting the somatic pathology, with the final aim of providing an effective and timely treatment of these global disorders.
Human Molecular Genetics 01/2014; 23(12). DOI:10.1093/hmg/ddu034 · 6.39 Impact Factor
"However, large vector doses are needed to achieve therapeutic benefits. The requirements for sufficient amounts of the vector pose a production challenge, as well as the risk of initiating the host immune response to the vector , , . More specifically, recombinant vectors based on AAV2 serotype were initially used in a clinical trial for the potential gene therapy of hemophilia B, but in this trial, therapeutic level of expression of human Factor IX (hF.IX) was not achieved at lower vector doses, and at higher vector doses, the therapeutic level of expression of hF.IX was short-lived due to a cytotoxic T cell (CTL) response against AAV2 capsids , , . "
[Show abstract][Hide abstract] ABSTRACT: The ubiquitin-proteasome pathway plays a critical role in the intracellular trafficking of AAV2 vectors, and phosphorylation of certain surface-exposed amino acid residues on the capsid provides the primary signal for ubiquitination. Removal of several critical tyrosine (Y) and serine (S) residues on the AAV2 capsid has been shown to significantly increase transduction efficiency compared with the wild-type (WT) vectors. In the present study, site-directed mutagenesis of each of the 17 surface-exposed threonine (T) residues was conducted, and the transduction efficiency of four of these mutants, T455V, T491V, T550V, and T659V, was observed to increase up to 4-fold in human HEK293 cells in vitro. The most critical Y, S, and T mutations were subsequently combined, and the quadruple-mutant (Y444+500+730F+T491V) AAV2 vector was identified as the most efficient. This vector increased the transduction efficiency ∼24-fold over the WT AAV2 vector, and ∼2-3-fold over the previously described triple-mutant (Y444+500+730F) vector in a murine hepatocyte cell line, H2.35, in vitro. Similar results were obtained in murine hepatocytes in vivo following tail vein injection of the Y444+500+730F+T491V scAAV2 vector, and whole-body bioluminescence imaging of C57BL/6 mice. The increase in the transduction efficiency of the Y-T quadruple-mutant over that of the Y triple-mutant correlated with an improved nuclear translocation of the vectors, which exceeded 90%. These observations suggest that further optimization of the AAV2 capsid by targeting amino acid residues involved in phosphorylation may not be possible. This study has thus led to the generation of a novel Y444+500+730F+T491V quadruple-mutant AAV2 vector with potential for use in liver-directed human gene therapy.
PLoS ONE 03/2013; 8(3):e59142. DOI:10.1371/journal.pone.0059142 · 3.23 Impact Factor
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