Cathryn Mah

Human fibroblast growth factor receptor 1 is a co-receptor for infection by adeno-associated virus 2

Adeno-associated virus 2 (AAV)-based vectors have gained attention as a potentially useful alternative to the more commonly used retroviral and adenoviral vectors for human gene therapy. Although AAV uses the ubiquitously expressed cell surface heparan sulfate proteoglycan (HSPG) as a receptor, the transduction efficiency of AAV vectors varies greatly in different cells and tissues in vitro and in vivo. We demonstrate here that cell surface expression of HSPG alone is insufficient for AAV infection, and that AAV also requires human fibroblast growth factor receptor 1 (FGFR1) as a co-receptor for successful viral entry into the host cell. We document that cells that do not express either HSPG or FGFR1 fail to bind AAV and, consequently, are resistant to infection by AAV. These non-permissive cells are successfully transduced by AAV vectors after stable transfections with cDNAs encoding the murine HSPG and the human FGFR1. Furthermore, AAV infection of permissive cells, known to express both FGFR1 and the epidermal growth factor receptor, is abrogated by treatment of cells with basic fibroblast growth factor, but not with epidermal growth factor. The identification of FGFR1 as a co-receptor for AAV should provide new insights not only into its role in the life cycle of AAV, but also in the optimal use of AAV vectors in human gene therapy.

Next generation of adeno-associated virus 2 vectors: Point mutations in tyrosines lead to high-efficiency transduction at lower doses

Recombinant adeno-associated virus 2 (AAV2) vectors are in use in several Phase I/II clinical trials, but relatively large vector doses are needed to achieve therapeutic benefits. Large vector doses also trigger an immune response as a significant fraction of the vectors fails to traffic efficiently to the nucleus and is targeted for degradation by the host cell proteasome machinery. We have reported that epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK) signaling negatively affects transduction by AAV2 vectors by impairing nuclear transport of the vectors. We have also observed that EGFR-PTK can phosphorylate AAV2 capsids at tyrosine residues. Tyrosine-phosphorylated AAV2 vectors enter cells efficiently but fail to transduce effectively, in part because of ubiquitination of AAV capsids followed by proteasome-mediated degradation. We reasoned that mutations of the surface-exposed tyrosine residues might allow the vectors to evade phosphorylation and subsequent ubiquitination and, thus, prevent proteasome-mediated degradation. Here, we document that site-directed mutagenesis of surface-exposed tyrosine residues leads to production of vectors that transduce HeLa cells ≈10-fold more efficiently in vitro and murine hepatocytes nearly 30-fold more efficiently in vivo at a log lower vector dose. Therapeutic levels of human Factor IX (F.IX) are also produced at an ≈10-fold reduced vector dose. The increased transduction efficiency of tyrosine-mutant vectors is due to lack of capsid ubiquitination and improved intracellular trafficking to the nucleus. These studies have led to the development of AAV vectors that are capable of high-efficiency transduction at lower doses, which has important implications in their use in human gene therapy.

Recombinant Adeno-Associated Virus Serotype 9 Leads to Preferential Cardiac Transduction In Vivo

Heart disease is often the end result of inherited genetic defects, which may potentially be treatable using a gene-transfer approach. Recombinant adeno-associated virus (rAAV)-mediated gene delivery has emerged as a realistic method for the treatment of such disorders. Here, we demonstrate and compare the natural affinity of specific AAV serotype capsids for transduction of cardiac tissue. We compared the previously accepted optimal rAAV serotype for transduction of skeletal muscle, rAAV2/1, with rAAV2/8 and the newer rAAV2/9 vectors carrying the CMV-lacZ construct in their respective abilities to transcend vasculature and transduce myocardium following intravenous delivery of 1×1011 vector genomes in neonatal mice. We found that both rAAV2/8 and rAAV2/9 are able to transduce myocardium at ≈20- and 200-fold (respectively) higher levels than rAAV2/1. Biodistribution analysis revealed that rAAV2/9 and rAAV2/8 demonstrate similar behavior in extracardiac tissue. Vector genome quantification showed an increase in genome copy numbers in cardiac tissue for several weeks following administration, which corresponds to expression data. In addition, we intravenously administered 1×1011 vector genomes of rAAV2/9-CMV-lacZ into adult mice and achieved an expression biodistribution profile similar to that found following delivery to newborns. Although higher doses of virus will be necessary to approach those levels observed following neonatal injections, adult myocardium is also readily transduced by rAAV2/9. Finally, we have demonstrated physiological disease correction by AAV9 gene transfer in a mouse model of Pompe disease via ECG tracings and that intravenous delivery of the same vector preferentially transduces cardiac tissue in nonhuman primates.

Impaired intracellular trafficking of adeno-associated virus type 2 vectors limits efficient transduction of murine fibroblasts

ABSTRACT Although adeno-associated virus type 2 (AAV) has gained attention as a potentially useful alternative to the more commonly used retrovirus- and adenovirus-based vectors for human gene therapy, efficient gene transfer and transgene expression by AAV vectors require that the following two obstacles be overcome. First, the target cell must express the receptor and the coreceptor for AAV infection, and second, the cell must allow for viral second-strand DNA synthesis. We now describe a third obstacle, impaired intracellular trafficking of AAV to the nucleus, which results in the lack of transgene expression in murine fibroblasts which do express the AAV receptor and the coreceptor and which are permissive for viral second-strand DNA synthesis. We document that AAV vectors bind efficiently and gain entry successfully into NIH 3T3 cells, but trafficking into the nucleus is significantly impaired in these cells. In contrast, viral trafficking to the nucleus in cells known to be efficiently transduced by AAV vectors is both rapid and efficient. The demonstration of yet another obstacle in AAV-mediated gene transfer has implications for the optimal use of these vectors in human gene therapy.

Tyrosine phosphorylation of AAV2 vectors and its consequences on viral intracellular trafficking and transgene expression

We have documented that epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK) signaling negatively affects intracellular trafficking and transduction efficiency of recombinant adeno-associated virus 2 (AAV2) vectors. Specifically, inhibition of EGFR-PTK signaling leads to decreased ubiquitination of AAV2 capsid proteins, which in turn, facilitates viral nuclear transport by limiting proteasome-mediated degradation of AAV2 vectors. In the present studies, we observed that AAV capsids can indeed be phosphorylated at tyrosine residues by EGFR-PTK in in vitro phosphorylation assays and that phosphorylated AAV capsids retain their structural integrity. However, although phosphorylated AAV vectors enter cells as efficiently as their unphosphorylated counterparts, their transduction efficiency is significantly reduced. This reduction is not due to impaired viral second-strand DNA synthesis since transduction efficiency of both single-stranded AAV (ssAAV) and self-complementary AAV (scAAV) vectors is decreased by approximately 68% and approximately 74%, respectively. We also observed that intracellular trafficking of tyrosine-phosphorylated AAV vectors from cytoplasm to nucleus is significantly decreased, which results from ubiquitination of AAV capsids followed by proteasome-mediated degradation, although downstream consequences of capsid ubiquitination may also be affected by tyrosine-phosphorylation. These studies provide new insights into the role of tyrosine-phosphorylation of AAV capsids in various steps in the virus life cycle, which has implications in the optimal use of recombinant AAV vectors in human gene therapy.

Neural deficits contribute to respiratory insufficiency in Pompe disease

Pompe disease is a severe form of muscular dystrophy due to glycogen accumulation in all tissues, especially striated muscle. Disease severity is directly related to the deficiency of acid α-glucosidase (GAA), which degrades glycogen in the lysosome. Respiratory dysfunction is a hallmark of the disease, muscle weakness has been viewed as the underlying cause, and the possibility of an associated neural contribution has not been evaluated previously. Therefore, we examined behavioral and neurophysiological aspects of breathing in 2 animal models of Pompe disease—the Gaa−/− mouse and a transgenic line (MTP) expressing GAA only in skeletal muscle, as well as a detailed analysis of the CNS in a Pompe disease patient. Glycogen content was elevated in the Gaa−/− mouse cervical spinal cord. Retrograde labeling of phrenic motoneurons showed significantly greater soma size in Gaa−/− mice vs. isogenic controls, and glycogen was observed in Gaa−/− phrenic motoneurons. Ventilation, assessed via plethysmography, was attenuated during quiet breathing and hypercapnic challenge in Gaa−/− mice (6 to >21 months of age) vs. controls. We confirmed that MTP mice had normal diaphragmatic contractile properties; however, MTP mice had ventilation similar to the Gaa−/− mice during quiet breathing. Neurophysiological recordings indicated that efferent phrenic nerve inspiratory burst amplitudes were substantially lower in Gaa−/− and MTP mice vs. controls. In human samples, we demonstrated similar pathology in the cervical spinal cord and greater accumulation of glycogen in spinal cord compared with brain. We conclude that neural output to the diaphragm is deficient in Gaa−/− mice, and therapies targeting muscle alone may be ineffective in Pompe disease.

Virus-Based Gene Delivery Systems

Within the past decade, gene therapy strategies have come to the forefront of novel therapeutics. Tremendous advances in vector technology along with deeper understandings of vector biology and the molecular mechanisms of disease have significantly advanced the field of human gene therapy. This manuscript will discuss the viral-based subset of current gene transfer vectors. In particular, the most established viral vectors to date, including parvovirus, adenovirus, retro-virus, lentivirus, and herpesvirus-based vectors, are described, as well as the current innovative improvements being made to each. From past experience, it has become evident that in addition to optimising the vectors in terms of transgene expression, minimising vector-related immunology, and vector production, methods of vector delivery resulting in optimum vector transduction of target cells need to be established. This review will also illustrate several current improved physical delivery systems for optimal vector administration.

Pompe disease gene therapy

Pompe disease is an autosomal recessive metabolic myopathy caused by the deficiency of the lysosomal enzyme acid alpha-glucosidase and results in cellular lysosomal and cytoplasmic glycogen accumulation. A wide spectrum of disease exists from hypotonia and severe cardiac hypertrophy in the first few months of life due to severe mutations to a milder form with the onset of symptoms in adulthood. In either condition, the involvement of several systems leads to progressive weakness and disability. In early-onset severe cases, the natural history is characteristically cardiorespiratory failure and death in the first year of life. Since the advent of enzyme replacement therapy (ERT), the clinical outcomes have improved. However, it has become apparent that a new natural history is being defined in which some patients have substantial improvement following ERT, while others develop chronic disability reminiscent of the late-onset disease. In order to improve on the current clinical outcomes in Pompe patients with diminished clinical response to ERT, we sought to address the cause and potential for the treatment of disease manifestations which are not amenable to ERT. In this review, we will focus on the preclinical studies that are relevant to the development of a gene therapy strategy for Pompe disease, and have led to the first clinical trial of recombinant adeno-associated virus-mediated gene-based therapy for Pompe disease. We will cover the preliminary laboratory studies and rationale for a clinical trial, which is based on the treatment of the high rate of respiratory failure in the early-onset patients receiving ERT.

Induction of tolerance to factor VIII by transient co-administration with rapamycin.

See also Miao CH. Tilt balance towards regulation: evolving new strategy for treatment of hemophilia inhibitors. This issue, pp 1521–3.DOI:10.1111/j.1538‐7836.2011.04351.x.

Characterization of a Transgenic Short Hairpin RNA-Induced Murine Model of Tafazzin Deficiency

Barths syndrome (BTHS) is an X-linked mitochondrial disease that is due to a mutation in the Tafazzin (TAZ) gene. Based on sequence homology, TAZ has been characterized as an acyltransferase involved in the metabolism of cardiolipin (CL), a unique phospholipid almost exclusively located in the mitochondrial inner membrane. Yeast, Drosophila, and zebrafish models have been invaluable in elucidating the role of TAZ in BTHS, but until recently a mammalian model to study the disease has been lacking. Based on in vitro evidence of RNA-mediated TAZ depletion, an inducible short hairpin RNA (shRNA)-mediated TAZ knockdown (TAZKD) mouse model has been developed (TaconicArtemis GmbH, Cologne, Germany), and herein we describe the assessment of this mouse line as a model of BTHS. Upon induction of the TAZ-specific shRNA in vivo, transgenic mouse TAZ mRNA levels were reduced by >89% in cardiac and skeletal muscle. TAZ deficiency led to the absence of tetralineoyl-CL and accumulation of monolyso-CL in cardiac muscle. Furthermore, mitochondrial morphology from cardiac and skeletal muscle was altered. Skeletal muscle mitochondria demonstrated disrupted cristae, and cardiac mitochondria were significantly enlarged and displace neighboring myofibrils. Physiological measurements demonstrated a reduction in isometric contractile strength of the soleus and a reduction in cardiac left ventricular ejection fraction of TAZKD mice compared with control animals. Therefore, the inducible TAZ-deficient model exhibits some of the molecular and clinical characteristics of BTHS patients and may ultimately help to improve our understanding of BTHS-related cardioskeletal myopathy as well as serve as an important tool in developing therapeutic strategies for BTHS.