Biomedical subjects

Nils Loewen

Publications and source records attributed to Nils Loewen.

8 recordsLinked to original sources

PubMed2006

Perfusion of his-tagged eukaryotic myocilin increases outflow resistance in human anterior segments in the presence of aqueous humor.

PURPOSE: A previous study by the authors has shown that recombinant myocilin purified from a prokaryotic expression system increases outflow resistance in cultured human anterior segments. The present study was performed to determine whether full-length myocilin purified from a human trabecular meshwork cell expression system alters outflow resistance after infusion into human anterior segments. METHODS: A feline immunodeficiency virus vector encoding both full-length myocilin (amino acids 1-503 fused to C-terminal V5 and six-histidine epitopes) and puromycin resistance was used to transduce a transformed trabecular meshwork cell line (TM5). Stably expressing cells were selected with puromycin. Recombinant myocilin was purified from the media using nickel ion affinity chromatography. Control purifications were performed on media from parental TM5 cells. Anterior segments of human eyes were placed in organ culture and perfused with either Dulbecco's modified Eagle's medium (DMEM) or DMEM supplemented with 50% porcine aqueous humor. One eye received an anterior chamber exchange with recombinant myocilin (2 microg/mL), whereas the fellow eye received an equal volume of control. Immunohistochemistry was performed with anti-myocilin and anti-V5 antibodies. Native polyacrylamide gel electrophoresis was used to analyze myocilin complex formation in porcine aqueous humor. RESULTS: Recombinant myocilin in porcine aqueous humor increased outflow resistance in cultured human anterior segments (91% +/- 68% [mean +/- SD] versus 18% +/- 31% in fellow control eye; n = 9, P = 0.004). Maximum outflow resistance was obtained 5 to 17 hours after infusion and remained above baseline for >3 days. Recombinant myocilin also increased outflow resistance in eyes incubated in DMEM, but only if myocilin was preincubated with porcine aqueous humor (78% +/- 77% when preincubated in DMEM containing porcine aqueous humor versus 13% +/- 15% when preincubated with DMEM alone, n = 6, P = 0.03). Recombinant myocilin appears to form a complex in porcine aqueous humor with a heat-labile protein(s). Immunohistochemistry revealed the presence of myocilin in the juxtacanalicular region of the trabecular meshwork. CONCLUSIONS: Myocilin purified from human trabecular meshwork cells increased outflow resistance in cultured human anterior segments, but only after incubation with porcine aqueous humor. Recombinant myocilin appears to form a complex in porcine aqueous humor that enables it to bind specifically within the trabecular meshwork.

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PubMed2005

Lentiviral vectors.

We review the use of lentiviral vectors in current human gene therapy applications that involve genetic modification of nondividing tissues with integrated transgenes. Safety issues, including insertional mutagenesis and replication-competent retroviruses, are discussed. Innate cellular defenses against retroviruses and their implications for human gene therapy with different lentiviral vectors are also addressed.

Genetic Therapy↗

PubMed2004

Long-term retinal transgene expression with FIV versus adenoviral vectors.

PURPOSE: Gene therapy for chronic retinal diseases will require long-term expression of therapeutic transgenes. Lentiviral and adenoviral (Ad) vectors are gene delivery systems with markedly different properties. Lentiviral vectors require integration into the host genome, which facilitates long-term expression, while Ad vectors remain episomal. We compared time course, location, and extent of transgene expression from replication-deficient feline immunodeficiency virus (FIV) vectors and Ad vectors in neonatal rat retina. METHODS: A dose-response study was conducted to determine the optimal subretinal dose for comparison of FIV and Ad vectors with an internal cassette expressing beta-galactosidase under transcriptional control of the CMV immediate-early gene promoter/enhancer. Forty-two five-day old Sprague-Dawley rats received subretinal injections of 2 microl containing 2x10(3) transducing units (TU, n=14), 2x10(4) TU (n=14) or 2x10(5) TU (n=14) of FIV vector (right eye) and Ad vector (left eye). Expression was evaluated 48 h after transduction. In the subsequent long-term expression study, 60 five-day old rats received a subretinal injection of 2x10(5) TU FIV vector (right eye) and Ad vector (left eye). Ten pairs of eyes were analyzed at 1 week, 1 month, 3 months, 6 months, 12 months, and the remainder at 16 months. Eye cups were evaluated in a masked manner for extent of beta-galactosidase expression (graded 0-5) by whole mount microscopy and by cross sectional histology. RESULTS: In the dose-response study, 2x10(5) TU resulted in consistent, widespread retinal transduction with both vectors and was selected as the dose for the subsequent study. In the long-term expression study, FIV vector resulted in a higher grade of expression than Ad at multiple single time points and produced higher overall expression when data from all eyes across the entire 16 month study were analyzed (p=0.01). Retinal expression was present at 16 months with both vectors. beta-galactosidase expression was limited to the retinal pigment epithelium (RPE) until the first month, but later was also found to a lesser extent in neurosensory retina with each vector. In contrast to FIV, most Ad injected eyes showed signs of focal accumulation of macrophage-like cells with disrupted retinal architecture. CONCLUSIONS: Both FIV and Ad vectors result in long-term transgene expression in RPE after subretinal injection. FIV vectors show more promise than Ad as delivery systems for retinal diseases since they transduce greater areas of RPE, result in less cellular infiltrate, and cause less disruption of retinal architecture. The persistent expression at 16 months of follow-up suggests that these lentiviral vectors are useful for gene therapy of chronic retinal diseases.

Adenoviridae↗

PubMed2004

Unintegrated lentivirus DNA persistence and accessibility to expression in nondividing cells: analysis with class I integrase mutants.

The circumstances under which unintegrated lentivirus DNA can persist and be a functional template for transcription and protein expression are not clear. We constructed and validated the first class I (nonpleiotropic) integrase (IN) mutants for a non-human lentivirus (feline immunodeficiency virus [FIV]) and analyzed both these and known class I human immunodeficiency virus type 1 IN mutants. The FIV IN mutants (D66V and D66V/D118A) had class I properties: Gag/Pol precursor expression, proteolytic processing, particle formation, and reverse transcriptase (RT) production were normal, while the transduction of dividing fibroblasts was prevented and integration was blocked. When injected into rat retinas, the wild-type (WT) vector produced extensive, persistent transgene expression, compared with only rare positive neuronal cells for the IN mutant vector. In contrast, both WT and mutant vectors produced entirely equivalent, effective transduction levels of primary rat neurons (retinal ganglion cells). By testing the hypothesis that the unexpected retinal neuron transduction was related to cell cycle status, we found that when fibroblasts were growth arrested, transduction and internally promoted transgene expression were not inhibited at all by the class I FIV or HIV-1 IN mutations. Cells were then transduced under aphidicolin arrest and were released from the block 48 h later. Vector expression was stable and durable during repeated passaging in WT vector-transduced cells, while the release of cells transduced with equivalent RT units of class I IN mutant FIV or HIV vector resulted in a steady decline of expression, from 97 to 0% of cells by day 10. Southern blot and PCR analyses showed a lack of integration, irrespective of cell cycle, for the class I mutants and an increase in one- and two-long terminal repeat circular and linear unintegrated DNAs in growth-arrested cells. We conclude that if cell division is prevented, unintegrated FIV and HIV-1 vector DNAs can produce high-level internally promoted transgene expression equivalent to WT vectors. The expression correlates with the unintegrated DNA levels. These observations may facilitate the study of the roles of IN and other preintegration complex components in preintegration phases of infection by (i) providing an alternative way to monitor unintegrated nuclear cDNA forms, (ii) restricting ascertainment to the transcriptionally functional subset of unintegrated DNA, (iii) enabling analysis in individual, nondividing cells, and (iv) uncoupling other potential functions of IN from integration.

Amino Acid Sequence↗

PubMed2004

Long-term, targeted genetic modification of the aqueous humor outflow tract coupled with noninvasive imaging of gene expression in vivo.

PURPOSE: To address a problem impeding research into glaucoma-associated genetic mutations and glaucoma gene therapy and achieve permanent, targeted transgene expression in the trabecular meshwork (TM). Lentiviral vectors are known to transduce human donor eye TM ex vivo, but efficacy in vivo has not been shown. More generally in the field of gene therapy, the authors hypothesized that distinctive properties of the intraocular aqueous circulation could facilitate solving problems of accessibility, targeting, and scale that have hindered realization of gene therapy in other settings. METHODS: A domestic cat model was developed in which long-term in vivo studies were performed. After dose-response studies in primary human TM cells, 19 cats received anterior chamber (AC) injections of stepped doses (10(6)-10(8) transduction units) of lentiviral vectors encoding different marker transgenes (beta-galactosidase, Aequorea victoria green fluorescent protein [GFP], or Renilla reniformis GFP). Animals were monitored serially for transgene expression and IOP. RESULTS: High-grade, stable transgene expression in the TM was achieved and monitored noninvasively over time in living animals. Extensive expression resulted after a single transcorneal injection, persisted for at least 10 months (time of death in the present studies), and was targeted to the TM. The initial IOP did not differ significantly from the IOP at the end of the study (P = 0.4). Aequorea GFP was superior to Renilla GFP. Vectors were effective enough to cause GFP-specific overexpression cytotoxicity at the highest dose, which was solved by dose reduction. CONCLUSIONS: High-grade transgene expression in this large-animal model persisted stably for at least 10 months after a single transcorneal lentiviral vector injection, was highly targeted, and could be monitored serially and noninvasively in living animals. These studies provide a basis for developing realistic disease models and administering glaucoma gene therapy.

Animals↗

PubMed2003

Comparison of wild-type and class I integrase mutant-FIV vectors in retina demonstrates sustained expression of integrated transgenes in retinal pigment epithelium.

BACKGROUND: In neonatal and adult rodent retina, substantial lentiviral vector expression has been detected primarily in retinal pigment epithelium (RPE), except in very young animals (2-5 days post-natal). In non-retinal tissues, studies of lentiviral vectors have utilized various controls. Among the most stringent are class I integrase mutants, which selectively block the integration reaction while leaving all other gag/pol-encoded functions intact. For HIV-1 vectors injected into brain, these have been used to simultaneously control for pseudotransduction and verify that long-term expression requires integration. Such experiments compare particles that differ only in a single amino acid within a single enzyme that forms a very small molar fraction of the virion. Class I integrase mutants have not been described for feline immunodeficiency virus (FIV) integrase, or tested in the eye for any lentiviral vector. METHODS: We compared subretinally and intravitreally injected FIV vectors and followed animals for up to 7 months, a duration that exceeds prior studies. We also compared the wild-type (WT) vector with one incorporating a single class I amino acid mutation in FIV integrase (D66V). A mock vector (packaging construct absent) was an alternative control. All vectors were vesicular stomatitis virus glycoprotein G (VSV-G)-pseudotyped and were injected on day 7 of life. One group of animals received either subretinal or intravitreal injections of WT vector in the right eyes. Control left eyes were injected with mock vector. These animals were sacrificed at 2 or 7 days post-injection. A second group received subretinal injections of either WT vector or equivalent D66V vector (reverse transcriptase-normalized to WT), and were analyzed after 2, 3 and 7 months. All eyes were scored for marker gene (beta-galactosidase) expression by an observer blinded to vector assignments. RESULTS: Subretinal FIV vector injections were much more effective than intravitreal injections. The RPE was the principal retinal layer transduced by the WT vector, and at least 50% of the area of the retina expressed the marker gene at 3 and 7 months. Occasional cells in inner retinal layers also expressed beta-galactosidase at these time points. The sustained retinal expression produced by subretinally injected vector was blocked by the D66V mutation. CONCLUSIONS: These results show that class I integrase mutant FIV vectors are useful control vectors, and that VSV-G-pseudotyped FIV vectors produce extensive retinal expression for at least 215 days, the longest duration yet reported for lentiviral vectors in retina. Transgene expression is mostly restricted to RPE after post-natal day 7 in rats, suggesting that FIV vectors could be used to target RPE for gene therapy.

Age Factors↗

PubMed2003

FIV Vectors.

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Animals↗

PubMed2002

Preservation of aqueous outflow facility after second-generation FIV vector-mediated expression of marker genes in anterior segments of human eyes.

PURPOSE: Feline immunodeficiency virus (FIV)-based lentiviral vectors produce effective genetic modification of the trabecular meshwork (TM) of human eyes in organ-perfusion culture, resulting in high-level expression of a beta-galactosidase marker gene (lacZ) without loss of TM cellularity or architecture. However, effects on aqueous outflow physiology have not been determined, and the ability to monitor FIV vector transgene expression in living TM in situ has not been established. In the current study, transgene expression and outflow facility were evaluated in perfused human anterior segments after FIV vector transduction of lacZ or of a marker gene that can be monitored noninvasively, enhanced green fluorescent protein (eGFP). METHODS: Second-generation FIV vectors were made with a protocol for scaled-up production that requires 10 times less input DNA and allows simplified concentration. One vector encodes beta-galactosidase (vector CT26), and the other (bicistronic) encodes eGFP and neomycin phosphotransferase (vector GiNWF). Three pairs of eyes were injected with 1 x 10(8) transducing units (TU) of CT26 in the right eye and with a control (mock lacZ) vector in the left eye. Three others were injected with 1 x 10(8) TU GiNWF in the right eye only, with the left eye serving as an uninjected control. Intraocular pressure was recorded and transduction efficiency was determined. RESULTS: The modified protocol produced high-titer FIV vectors, and coordinate expression of marker genes was observed with the bicistronic vector. In human eyes, the eGFP and lacZ vectors transduced 79% +/- 15% and 82% +/- 4% of TM cells, respectively, without cell loss compared with control eyes. Transduction and marker gene expression caused a transient decrease of outflow facility (30% +/- 22%, P = 0.02), which resolved after 48 to 72 hours. CONCLUSIONS: FIV vectors produce high-level expression of eGFP in the TM of the cultured human eye, with transduction efficiency similar to that obtained with beta-galactosidase vectors. Transduction and expression of these marker genes results in small and transient changes in outflow facility, suggesting suitability of this class of vectors for glaucoma gene therapy.

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