Manij Patel

Influenza M2 envelope protein augments avian influenza hemagglutinin pseudotyping of lentiviral vectors

Lentivirus-based gene transfer has the potential to efficiently deliver DNA-based therapies into non-dividing epithelial cells of the airway for the treatment of lung diseases such as cystic fibrosis. However, significant barriers both to lung-specific gene transfer and to production of lentivirus vectors must be overcome before these vectors can be routinely used for applications to the lung. In this study, we investigated whether the ability to produce lentiviral vectors pseudotyped with fowl plague virus hemagglutinin (HA) could be improved by co-expression of influenza virus M2 in vector-producing cells. We found that M2 expression led to a 10–30-fold increase in production of HA-pseudotyped lentivirus vectors based upon equine infectious anemia virus (EIAV) or human immunodeficiency virus type 1 (HIV-1). Experiments using the M2 inhibitor amantadine and a drug-resistant mutant of M2 established that the ion channel activity of M2 was important for M2-dependent augmentation of vector production. Furthermore, the neuraminidase activity necessary for particle release from producer cells could also be incorporated into producer cells by co-expression of influenza NA cDNA. Lentiviral vectors pseudotyped with influenza envelope proteins were able to efficiently transduce via the apical membrane of polarized mouse tracheal cultures in vitro as well as mouse tracheal epithelia in vivo.

Analysis of gene transfer and expression in skeletal muscle using enhanced EIAV lentivirus vectors

Skeletal muscle is an attractive target tissue for gene therapy involving both muscle and nonmuscle disorders. HIV-1-based vectors transduce mature skeletal muscle; however, the use of these vectors for human gene therapy may be limited by biosafety concerns. In this study, we investigated gene transfer using lentivirus vectors based on the equine infectious anemia virus (EIAV) in skeletal muscle in vitro and in vivo. EIAV vectors transduce proliferating and differentiating C2C12 mouse muscle cells; furthermore, the addition of the woodchuck hepatitis posttranscriptional element to EIAV vectors markedly increases gene expression in these cells. A single injection of EIAV vectors into skeletal muscle of adult mice led to detectable gene marking and gene expression for the duration of the 3-month study. Use of a second-generation EIAV self-inactivating vector (E-SIN) increased transduction in muscle cells in vitro, and injection of E-SIN vectors into skeletal muscle resulted in increased gene marking and gene expression compared to first-generation EIAV vectors.

High efficiency gene transfer to airways of mice using influenza hemagglutinin pseudotyped lentiviral vectors

A limitation to efficient lentivirus‐mediated airway gene transfer is the lack of receptors to commonly used viral envelopes on the luminal surface of airway epithelia. The use of viral envelopes with natural tropism to the airway could be useful for overcoming this limitation.

Analysis of neuronal proliferation, migration and differentiation in the postnatal brain using equine infectious anemia virus-based lentiviral vectors

Ongoing neurogenesis in discrete sectors of the adult central nervous system depends on the mitotic activity of an elusive population of adult stem cells. The existence of adult neural stem cells provides an alternative approach to transplantation of embryonic stem cells in cell-based therapies. Owing to the limited intrinsic fate of adult stem cells and inhibitory nature of the adult brain for neurogenesis, accommodation for circuit replacement in the brain will require genetic and epigenetic manipulation. Here, we show that a replication-incompetent Equine Infectious Anemia Virus (EIAV) is highly suitable for stable and persistent gene transfer to adult neural stem cells. The transduced regions were free of long-lasting neuroimmune responses to EIAV. Transduction in the subventricular zone was specific to the stem cell niche, but spared the progeny of adult neural stem cells that includes transit amplifying progenitors (TAPs) and migrating neuroblasts. With time, EIAV-transduced stem cells passed on the transgene to TAPs and migrating neuroblasts, which ultimately differentiated into neurons in the olfactory bulbs. We show that EIAV is highly suitable for discovery and assessment of mechanisms that regulate proliferation, migration and differentiation in the postnatal brain.

Restoring ciliary function to differentiated Primary Ciliary Dyskinesia cells with a lentiviral vector

Primary ciliary dyskinesia (PCD) is a genetically heterogenous autosomal recessive disease in which mutations disrupt ciliary function, leading to impaired mucociliary clearance and life-long lung disease. Mouse tracheal cells with a targeted deletion in the axonemal dynein intermediate chain 1 (Dnaic1) gene differentiate normally in culture but lack ciliary activity. Gene transfer to undifferentiated cultures of mouse Dnaic1−/− cells with a lentiviral vector pseudotyped with avian influenza hemagglutinin restored Dnaic1 expression and ciliary activity. Importantly, apical treatment of well-differentiated cultures of mouse Dnaic1−/− cells with lentiviral vector also restored ciliary activity, demonstrating successful gene transfer from the apical surface. Treatment of Dnaic1flox/flox mice expressing an estrogen-responsive Cre recombinase with different doses of tamoxifen indicated that restoration of ∼20% of ciliary activity may be sufficient to prevent the development of rhinosinusitis. However, although administration of a β-galactosidase-expressing vector into control mice demonstrated efficient gene transfer to the nasal epithelium, treatment of Dnaic1−/− mice resulted in a low level of gene transfer, demonstrating that the severe rhinitis present in these animals impedes gene transfer. The results demonstrate that gene replacement therapy may be a viable treatment option for PCD, but further improvements in the efficiency of gene transfer are necessary.

492. Characterization of the Apical Transduction of Well-Differentiated Mouse Tracheal Cultures by an Equine Lentivirus Pseudotyped with Avian Influenza Coat Proteins

The main goal in gene therapy for Cystic Fibrosis (CF) is to transfer the CFTR transgene to the ciliated cells of the upper airways via an easily accessible route and provide long-term therapeutic expression. The ideal route of administration would be lung lumenal instillation but this requires vector uptake from the apical surface of the epithelium. The lung lumen provides many layers of defense to airborne pathogens such as a thick mucus layer and glycocalyx. We addressed these issues by using coat proteins from an influenza virus, which is able to infect the adult lung, to pseudotype a lentiviral vector capable of genome integration and long-term transgene expression. Equine Infectious Anemia Virus (EIAV) based vectors were pseudotyped with coat proteins of Fowl Plague Virus (FPV), an avian Influenza, and we were able to obtain titers greater than 105 iu/ml. Furthermore, we were able to concentrate titers up to 1,000-fold by ultracentrifugation to final titers approaching 108 iu/ml.

503. Gene transfer strategies for pulmonary hypertension

Pulmonary hypertension may be a potential disease target for alveolar gene transfer by luminal delivery. We hypothesize that long-term expression of prostacyclin synthase (PGIS) cDNA can ameliorate the manifestations of pulmonary hypertension in mouse models. To test this hypothesis, we are exploring the utility of alveolar gene transfer mediated by luminal delivery of adenoassociated virus (AAV) and equine infectious anemia virus (EIAV) vectors in comparison to vascular (internal jugular vein) route of delivery in vivo. AAV (serotypes 1 and 5) and EIAV vectors pseudotyped with the vesicular stomatis virus (VSV) glycoprotein (G) were produced containing either reporter genes or the PGIS cDNA under control of the clara cell 10 kd (CC10), beta actin promoter with cytomegalovirus enhancer (CB), or murine U1a promoters. Vector (50 ul of 1012 vector genomes/ml AAV; 109 infectious units (IU)/ml EIAV) was instilled luminally (nasally or intratracheally) and compared to internal jugular vein (IJ) administration of the same dose of vec-tor in mice. The mice were euthanized and transgene expression assessed by biochemical assays and histochemistry. In initial studies, AAV5-CB human placental alkaline phosphatase (hPLAP) vectors and AAV5-U1a green fluorescent protein (GFP) vectors mediated more efficient gene transfer to alveolar epithelia than AAV1 vectors with the same reporter expression cassettes. Transgene expression was detected in multiple cell types by histochemical analysis following transduction with AAV vectors containing the CB and U1a promoters, whereas expression from an AAV vector with a CC10 promoter was more specific to alveolar type II and airway epithelial cells. Luminal gene transfer efficiency was similar to IJ vein administration for AAV1 vectors, but AAV5 vectors provided more efficient gene transfer following luminal (nasal) rather than IJ delivery. Both AAV1 and AAV5 vectors mediated reporter gene expression for more than 3 months following nasal administration when driven by CB and U1a promoters, but AAV1 vector-mediated expression was attenuated by 70% over a 60 day period, whereas AAV5-mediated re-porter gene expression was only attenuated by 30%. Expression from both vectors remained stable from 60–90 days after transduction. With regard to lentivectors, luminal (intratracheal) delivery of a VSV-G pseudotyped EIAV-lacZ vector exhibited inefficient gene transfer. However, in vivo luminal delivery of a VSV-G pseudotyped EIAV-PGIS vector mediated a 1.3 fold increase in 6-keto-PGF1alpha (the nonenzymatic hydrolysis product of prostacyclin) levels in serum and IJ delivery induced a 2.2-fold increase in serum 6-ketoPGF1alpha levels. These data suggest that vectors with potential for long-term expression can mediate reporter gene and therapeutic transgene expression following in vivo delivery by vascular and luminal delivery routes and offer hope for gene transfer approaches to pulmonary hypertension.