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Patrick L Sinn

Publications and source records attributed to Patrick L Sinn.

11 recordsLinked to original sources
PubMed2025

Lentiviral-mediated gene complementation rescues pathogenic ABCA3 variants.

The ATP-binding cassette subfamily A member 3 (ABCA3) protein on the limiting membrane of lamellar bodies in alveolar type 2 (AT2) cells transports phospholipids required for pulmonary surfactant assembly. ABCA3 deficiency results from biallelic pathogenic variants in ABCA3 and causes progressive neonatal respiratory failure or childhood interstitial lung disease (chILD). Supportive/compassionate care or lung transplantation are the only current definitive treatments for ABCA3 deficiency and progressive respiratory failure. Complementing dysfunctional ABCA3 by gene addition has therapeutic potential. Previous studies show that repairing or complementing ABCA3 in induced pluripotent stem cell (iPSC)-derived AT2 cells rescues lamellar body morphology and surfactant phospholipid composition. Pathogenic variants disrupt ABCA3 function through altered protein trafficking (type 1) or by impaired phospholipid transport (type 2) into lamellar bodies. Here we tested ABCA3 gene complementation using a human pulmonary epithelial cell line (A549) with a genomically silenced ABCA3 locus (ABCA3 KO). From this line, additional cell lines that stably express individual ABCA3 variant cDNA constructs from a single genomic locus were tested: L101P (type 1), E292V (type 2), E690K (type 2), or wild-type ABCA3. Lentiviral-mediated ABCA3 delivery to each cell line partially rescued localization to LAMP3+ vesicles, lamellar body-like structure morphology, and cell proliferation. A functional assay measuring NF-κB signaling suggested that ABCA3 complementation ameliorated aberrant inflammatory signaling in E292V or E690K (type 2) mutant lines, but not in L101P (type 1) or knockout lines. These studies highlight the therapeutic potential of gene addition as well as differences between ABCA3 pathogenic variants that may influence genetic therapy outcomes.

ABCA3 deficiency↗
PubMed2015

Ferret and pig models of cystic fibrosis: prospects and promise for gene therapy.

Large animal models of genetic diseases are rapidly becoming integral to biomedical research as technologies to manipulate the mammalian genome improve. The creation of cystic fibrosis (CF) ferrets and pigs is an example of such progress in animal modeling, with the disease phenotypes in the ferret and pig models more reflective of human CF disease than mouse models. The ferret and pig CF models also provide unique opportunities to develop and assess the effectiveness of gene and cell therapies to treat affected organs. In this review, we examine the organ disease phenotypes in these new CF models and the opportunities to test gene therapies at various stages of disease progression in affected organs. We then discuss the progress in developing recombinant replication-defective adenoviral, adeno-associated viral, and lentiviral vectors to target genes to the lung and pancreas in ferrets and pigs, the two most affected organs in CF. Through this review, we hope to convey the potential of these new animal models for developing CF gene and cell therapies.

Animals↗
PubMed2005

Viscoelastic gel formulations enhance airway epithelial gene transfer with viral vectors.

Advances in gene transfer to the conducting airways for the treatment of pulmonary diseases such as cystic fibrosis have identified several vector classes that transduce airway epithelia in vitro and in animal models. One barrier to epithelial gene transfer is the rapid removal of materials from the airway surface via mucociliary clearance. This host defense mechanism limits gene transfer efficiency to airway epithelial cells. Here we show that formulation of gene transfer vectors with viscoelastic gels provides longer epithelial residence time and increases vector-mediated gene transfer efficiency. Gene transfer with adenoviral, adeno-associated, and lentiviral vectors all significantly improved after formulation with viscoelastic gels designed to slow mucociliary clearance. Importantly, viscoelastic gel formulations enhanced vector transduction to the conducting airways, the desired treatment target for diseases such as cystic fibrosis.

Adenoviridae↗
PubMed2005

Inclusion of Jaagsiekte sheep retrovirus proviral elements markedly increases lentivirus vector pseudotyping efficiency.

Retroviral pseudotyping for gene transfer applications endeavors to alter vector tropism and maintain a suitable titer. We investigated the compatibility of the Jaagsiekte sheep retrovirus (JSRV) envelope glycoprotein with the feline immunodeficiency virus (FIV) vector. A construct consisting of the minimal JSRV env coding region expressed from a standard mammalian expression plasmid generated FIV vector titers of approximately 10(4) TU/ml following standard triple transfection, collection of supernatants, and concentration by centrifuge. Interestingly, retention of the native proviral 5' and 3' flanking regions surrounding the JSRV env resulted in exceptional titers of approximately 10(8) TU/ml following the same viral preparation. To discern the regions necessary to achieve this 10,000-fold increase in titer, additional constructs were designed and tested. Our results indicate that the enhanced vector titer correlates with an increase in steady-state levels of envelope RNA that results from a combination of RNA splicing and stability, leading to increased envelope protein production. Expression of four other glycoproteins in an expression plasmid retaining the enhancing elements from the JSRV proviral sequence increased FIV vector titers from 0- to 100-fold. These novel data demonstrate that optimization of the envelope expression construct can profoundly influence titers for lentivirus vectors.

Animals↗
PubMed2005

Gene transfer to respiratory epithelia with lentivirus pseudotyped with Jaagsiekte sheep retrovirus envelope glycoprotein.

A feline immunodeficiency virus (FIV)-based lentiviral vector was pseudotyped to identify envelope (env) glycoproteins that direct efficient gene transfer to pulmonary epithelia for the treatment or prevention of lung diseases. The envelope glycoprotein from the Jaagsiekte sheep retrovirus (JSRV) is a candidate under investigation. We utilized high titer FIV vector (>10(8) TU/ml) pseudotyped with the JSRV env glycoprotein (JSRVFIV) to study the transduction of polarized primary cultures of human airway epithelia and receptor/vector interactions. The reported receptor for JSRV, hyaluronidase 2 (HYAL2), is a GPI-linked protein. We expressed FLAG-tagged HYAL2 in polarized airway epithelia using an adenoviral vector and documented that the HYAL2 protein sorts predominantly to the apical surface. Of interest, the efficiency of gene transfer with apically applied JSRV-FIV was markedly less than FIV pseudotyped with VSV-G, even in Ad-HYAL2 complemented epithelia. The inefficient gene transfer with JSRV-FIV in HYAL2 complemented cells suggests that factors other than receptor abundance limit apical gene transfer efficiency with this envelope. JSRV-FIV transduced the distal lung epithelia of rabbits in vivo and transduced primary cultures of rabbit type II cells with 100-fold greater efficiency than primary cultures of rabbit tracheal cells. These data indicate that a lentivirus pseudotyped with the JSRV envelope glycoprotein transduces type II cells with greater efficiency than conducting airway epithelia and provides an example of glycoprotein-mediated cell-specific tropism within a tissue with a widely heterogeneous cell population.

Animals↗
PubMed2005

Persistent gene expression in mouse nasal epithelia following feline immunodeficiency virus-based vector gene transfer.

Gene transfer development for treatment or prevention of cystic fibrosis lung disease has been limited by the inability of vectors to efficiently and persistently transduce airway epithelia. Influenza A is an enveloped virus with natural lung tropism; however, pseudotyping feline immunodeficiency virus (FIV)-based lentiviral vector with the hemagglutinin envelope protein proved unsuccessful. Conversely, pseudotyping FIV with the envelope protein from influenza D (Thogoto virus GP75) resulted in titers of 10(6) transducing units (TU)/ml and conferred apical entry into well-differentiated human airway epithelial cells. Baculovirus GP64 envelope glycoproteins share sequence identity with influenza D GP75 envelope glycoproteins. Pseudotyping FIV with GP64 from three species of baculovirus resulted in titers of 10(7) to 10(9) TU/ml. Of note, GP64 from Autographa californica multicapsid nucleopolyhedrovirus resulted in high-titer FIV preparations (approximately 10(9) TU/ml) and conferred apical entry into polarized primary cultures of human airway epithelia. Using a luciferase reporter gene and bioluminescence imaging, we observed persistent gene expression from in vivo gene transfer in the mouse nose with A. californica GP64-pseudotyped FIV (AcGP64-FIV). Longitudinal bioluminescence analysis documented persistent expression in nasal epithelia for approximately 1 year without significant decline. According to histological analysis using a LacZ reporter gene, olfactory and respiratory epithelial cells were transduced. In addition, methylcellulose-formulated AcGP64-FIV transduced mouse nasal epithelia with much greater efficiency than similarly formulated vesicular stomatitis virus glycoprotein-pseudotyped FIV. These data suggest that AcGP64-FIV efficiently transduces and persistently expresses a transgene in nasal epithelia in the absence of agents that disrupt the cellular tight junction integrity.

Animals↗
PubMed2003

Lentivirus vectors pseudotyped with filoviral envelope glycoproteins transduce airway epithelia from the apical surface independently of folate receptor alpha.

The practical application of gene therapy as a treatment for cystic fibrosis is limited by poor gene transfer efficiency with vectors applied to the apical surface of airway epithelia. Recently, folate receptor alpha (FR alpha), a glycosylphosphatidylinositol-linked surface protein, was reported to be a cellular receptor for the filoviruses. We found that polarized human airway epithelia expressed abundant FR alpha on their apical surface. In an attempt to target these apical receptors, we pseudotyped feline immunodeficiency virus (FIV)-based vectors by using envelope glycoproteins (GPs) from the filoviruses Marburg virus and Ebola virus. Importantly, primary cultures of well-differentiated human airway epithelia were transduced when filovirus GP-pseudotyped FIV was applied to the apical surface. Furthermore, by deleting a heavily O-glycosylated extracellular domain of the Ebola GP, we improved the titer of concentrated vector severalfold. To investigate the folate receptor dependence of gene transfer with the filovirus pseudotypes, we compared gene transfer efficiency in immortalized airway epithelium cell lines and primary cultures. By utilizing phosphatidylinositol-specific phospholipase C (PI-PLC) treatment and FR alpha-blocking antibodies, we demonstrated FR alpha-dependent and -independent entry by filovirus glycoprotein-pseudotyped FIV-based vectors in airway epithelia. Of particular interest, entry independent of FR alpha was observed in primary cultures of human airway epithelia. Understanding viral vector binding and entry pathways is fundamental for developing cystic fibrosis gene therapy applications.

Animals↗
PubMed2002

In vivo gene transfer using a nonprimate lentiviral vector pseudotyped with Ross River Virus glycoproteins.

Vectors derived from lentiviruses provide a promising gene delivery system. We examined the in vivo gene transfer efficiency and tissue or cell tropism of a feline immunodeficiency virus (FIV)-based lentiviral vector pseudotyped with the glycoproteins from Ross River Virus (RRV). RRV glycoproteins were efficiently incorporated into FIV virions, generating preparations of FIV vector, which after concentration attain titers up to 1.5 x 10(8) TU/ml. After systemic administration, RRV-pseudotyped FIV vectors (RRV/FIV) predominantly transduced the liver of recipient mice. Transduction efficiency in the liver with the RRV/FIV was ca. 20-fold higher than that achieved with the vesicular stomatitis virus G protein (VSV-G) pseudotype. Moreover, in comparison to VSV-G, the RRV glycoproteins caused less cytotoxicity, as determined from the levels of glutamic pyruvic transaminase and glutamic oxalacetic transaminase in serum. Although hepatocytes were the main liver cell type transduced, nonhepatocytes (mainly Kupffer cells) were also transduced. The percentages of the transduced nonhepatocytes were comparable between RRV and VSV-G pseudotypes and did not correlate with the production of antibody against the transgene product. After injection into brain, RRV/FIV preferentially transduced neuroglial cells (astrocytes and oligodendrocytes). In contrast to the VSV-G protein that targets predominantly neurons, <10% of the brain cells transduced with the RRV pseudotyped vector were neurons. Finally, the gene transfer efficiencies of RRV/FIV after direct application to skeletal muscle or airway were also examined and, although transgene-expressing cells were detected, their proportions were low. Our data support the utility of RRV glycoprotein-pseudotyped FIV lentiviral vectors for hepatocyte- and neuroglia-related disease applications.

Animals↗
PubMed2002

Measles virus preferentially transduces the basolateral surface of well-differentiated human airway epithelia.

Measles virus (MV) is typically spread by aerosol droplets and enters via the respiratory tract. The progression of MV infection has been widely studied; yet, the pathway for virus entry in polarized human airway epithelia has not been investigated. Herein we report the use of a replication-competent Edmonston vaccine strain of MV expressing enhanced green fluorescent protein (MV-eGFP) to infect primary cultures of well-differentiated human airway epithelia. Previous studies with polarized Caco-2 cells (intestine-derived human epithelia) and MDCK cells (kidney-derived canine epithelia) demonstrated that MV primarily infected and exited the apical surface. In striking contrast, our results indicate that MV preferentially transduces human airway cells from the basolateral surface; however, virus release remains in an apical direction. When MV-eGFP was applied apically or basolaterally to primary cultures of airway epithelia, discrete foci of eGFP expression appeared and grew; however, the cell layer integrity was maintained for the duration of the study (7 days). Interestingly, utilizing immunohistochemistry and confocal microscopy, we observed widespread expression of the receptor for the vaccine strain of MV (CD46) at greatest abundance on the apical surface of the differentiated human airway epithelia as well as in human tracheal tissue sections. These data suggest that the progression of MV infection through the respiratory epithelium may involve pathways other than direct binding and entry through the apical surface of airway epithelia.

Antigens, CD↗